Embryonic stem cells and neural progenitor cells derived therefrom

ABSTRACT

The present invention relates to undifferentiated human embryonic stem cells, methods of cultivation and propagation and production of differentiated cells. In particular it relates to the production of human ES cells capable of yielding somatic differentiated cells in vitro, as well as committed progenitor cells such as neural progenitor cells capable of giving rise to mature somatic cells including neural cells and/or glial cells and uses thereof.  
     This invention provides methods that generate in vitro and in vivo models of controlled differentiation of ES cells towards the neural lineage. The model, and cells that are generated along the pathway of neural differentiation may be used for: the study of the cellular and molecular biology of human neural development, discovery of genes, growth factors, and differentiation factors that play a role in neural differentiation and regeneration, drug discovery and the development of screening assays for teratogenic, toxic and neuroprotective effects.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of U.S. application Ser. No.09/808,382 filed Mar. 14, 2001.

[0002] The present invention relates to undifferentiated human embryonicstem cells, methods of cultivation and propagation and production ofdifferentiated cells. In particular it relates to the production ofhuman ES cells capable of yielding somatic differentiated cells invitro, as well as committed progenitor cells such as neural progenitorcells capable of giving rise to mature somatic cells including neuralcells and/or glial cells and uses thereof.

INTRODUCTION

[0003] The production of human embryonic stem cells which can be eithermaintained in an undifferentiated state or directed to undergodifferentiation into extraembryonic or somatic lineages in vitro allowsfor the study of the cellular and molecular biology of early humandevelopment, functional genomics, generation of differentiated cellsfrom the stem cells for use in transplantation or drug screening anddrug discovery in vitro.

[0004] In general, stem cells are undifferentiated cells which can giverise to a succession of mature functional cells. For example, ahaematopoietic stem cell may give rise to any of the different types ofterminally differentiated blood cells. Embryonic stem (ES) cells arederived from the embryo and are pluripotent, thus possessing thecapability of developing into any organ, cell type or tissue type or, atleast potentially, into a complete embryo.

[0005] The development of mouse ES cells in 1981 (Evans and Kaufman,1981; Martin, 1981) provided the paradigm, and, much of the technology,for the development of human ES cells. Development of ES cells evolvedout of work on mouse teratocarcinomas, (tumours arising in the gonads ofa few inbred strains), which consist of a remarkable array of somatictissues juxtaposed together in a disorganised fashion. Classical work onteratocarcinomas established their origins from germ cells in mice, andprovided the concept of a stem cell (the embryonal carcinoma or EC cell)which could give rise to the multiple types of tissue found in thetumours (Kleinsmith and Pierce, 1964; review, Stevens, 1983). The fieldof teratocarcinoma research (review, Martin, 1980) expanded considerablyin the 70's, as the remarkable developmental capacity of the EC stemcell became apparent following the generation of chimaeric mice byblastocyst injection of EC cells, and investigators began to realise thepotential value of cultured cell lines from the tumours as models formammalian development. EC cells however had limitations. They oftencontained chromosomal abnormalities, and their ability to differentiateinto multiple tissue types was often limited.

[0006] Since teratocarcinomas could also be induced by graftingblastocysts to ectopic sites, it was reasoned that it might be possibleto derive pluripotential cell lines directly from blastocysts ratherthan from tumours, as performed in 1981 by Gail Martin and Martin Evansindependently. The result was a stable diploid cell line which couldgenerate every tissue of the adult body, including germ cells.Teratocarcinomas also develop spontaneously from primordial germ cellsin some mouse strains, or following transplantation of primordial germcells to ectopic sites, and in 1992 Brigid Hogan and her colleaguesreported the direct derivation of EG cells from mouse primordial germcells (Matsui et al., 1992). These EG cells have a developmentalcapacity very similar to ES cells.

[0007] Testicular teratocarcinomas occur spontaneously in humans, andpluripotential cell lines were also developed from these (review,Andrews, 1988). Two groups reported the derivation of cloned cell linesfrom human teratocarcinoma which could differentiate in vitro intoneurons and other cell types (Andrews et al., 1984, Thompson et al.,1984). Subsequently, cell lines were developed which could differentiateinto tissues representative of all three embryonic germ layers (Pera etal., 1989). As analysis of the properties of human EC cells proceeded,it became clear that they were always aneuploid, usually (though notalways) quite limited in their capacity for spontaneous differentiationinto somatic tissue, and different in phenotype from mouse ES or ECcells.

[0008] The properties of the pluripotent cell lines developed by Pera etal. (1989) are as follows:

[0009] Express SSEA-3, SSEA-4, TRA 1-60, GCTM-2, alkaline phosphatase,Oct-4

[0010] Grow as flat colonies with distinct cell borders

[0011] Differentiate into derivatives of all three embryonic germ layers

[0012] Feeder cell dependent (feeder cell effect on growth notreconstituted by conditioned medium from feeder cells or by feeder cellextracellular matrix)

[0013] Highly sensitive to dissociation to single cells, poor cloningefficiency even on a feeder cell layer

[0014] Do not respond to Leukemia Inhibitory Factor

[0015] These studies of human EC cells essentially defined the phenotypeof primate pluripotential stem cells.

[0016] Derivation of primate ES cells from the rhesus monkey blastocystand later from that of the marmoset (Thomson et al., 1995, 1996) hasbeen described. These primate cell lines were diploid, but otherwisethey closely resembled their nearest counterpart, the human EC cell. Theimplication of the monkey work and the work on human EC cells was that apluripotent stem cell, which would be rather different in phenotype froma mouse ES cell, could likely be derived from a human blastocyst.

[0017] Bongso and coworkers (1994) reported the short term culture andmaintenance of cells from human embryos fertilised in vitro. The cellsisolated by Bongso and coworkers had the morphology expected ofpluripotent stem cells, but these early studies did not employ feedercell support, and it was impossible to achieve long term maintenance ofthe cultures.

[0018] James Thomson and coworkers (1998) derived ES cells from surplusblastocysts donated by couples undergoing treatment for infertility. Themethodology used was not very different from that used 17 years earlierto derive mouse ES stem cells. The trophectoderm, thought to beinhibitory to ES cell establishment, was removed by immunosurgery, theinner cell mass was plated on to a mouse embryonic fibroblast feedercell layer, and following a brief period of attachment and expansion,the resulting outgrowth was disaggregated and replated onto anotherfeeder cell layer. There were no significant departures from mouse ESprotocols in the media or other aspects of the culture system and arelatively high success rate was achieved. The phenotype of the cellswas similar to that outlined above in the human EC studies of Pera etal.

[0019] In the studies of Thomson et al. on monkey and human ES cells,there was no evidence that the cells showed the capacity for somaticdifferentiation in vitro. Evidence for in vitro differentiation waslimited to expression of markers characteristic of trophoblast andendoderm formation (production of human chorionic gonadotrophin andalphafetoprotein). It is not possible to state whether the cells foundproducing alphafetoprotein represent extraembryonic (yolk sac) endodermor definitive (embryonic) endoderm though the former is far more likely.Thus an essential feature for any human ES cell line to be of practicaluse, namely the production of differentiated somatic cells in vitro asseen in previous studies of human EC cells, was not demonstrated in themonkey or human ES cell studies.

[0020] Much attention recently has been devoted to the potentialapplications of stem cells in biology and medicine, the properties ofpluripotentiality and immortality are unique to ES cells and enableinvestigators to approach many issues in human biology and medicine forthe first time. ES cells potentially can address the shortage of donortissue for use in transplantation procedures, particularly where noalternative culture system can support growth of the required committedstem cell. However, it must be noted that almost all of the wide rangingpotential applications of ES cell technology in human medicine-basicembryological research, functional genomics, growth factor and drugdiscovery, toxicology, and cell transplantation are based on theassumption that it will be possible to grow ES cells on a large scale,to introduce genetic modifications into them, and to direct theirdifferentiation. Present systems fall short of these goals, but thereare indications of progress to come. The identification of novel factorsdriving pluripotential stem cell growth or stem cell selection protocolsto eliminate the inhibitory influence of differentiated cells, bothoffer a way forward for expansion and cloning of human ES cells.

[0021] The mammalian nervous system is a derivative of the ectodermalgerm layer of the postimplantation embryo. During the process of axisformation, it is thought that inductive signals elaborated by severalregions of the embryo (the anterior visceral endoderm and the earlygastrula organiser) induce the pluripotent cells of the epiblast toassume an anterior neural fate (Beddington and Robertson, 1999). Themolecular identity of the factors elaborated by these tissues whichdirect neurogenesis is unknown, but there is strong evidence from lowervertebrates that antagonists of the Wnt and BMP families of signallingmolecules may be involved.

[0022] Embryonic stem cells are pluripotent cells which are thought tocorrespond to the epiblast of the periimplantation embryo. Mouse EScells are able to give rise to neural tissue in vitro eitherspontaneously or during embryoid body formation. The neural tissue oftenforms in these circumstances in amongst a mixture of a range of celltypes. Alteration of the conditions of culture, or subsequent selectionof neural cells from this mixture, has been used to produce relativelypure populations of neuronal cells from differentiating cultures of EScells (eg Li et al., 1998). These neuronal cells have been used inexperimental models to correct various deficits in animal model systems(review, Svendsen and Smith, 1999). The same has not yet been achievedwith human ES cell derived neurons, though neuronal cells have beenderived from human embryonal carcinoma cells which were induced todifferentiate using retinoic acid. These EC cells were subsequentlyshown to correct deficits in experimental models of CNS disease.

[0023] A suitable source of human ES derived neurons would be desirablesince their availability would provide real advantages for basic andapplied studies of CNS development and disease. Controlleddifferentiation of human ES cells into the neural lineage will allowexperimental dissection of the events during early development of thenervous system, and the identification of new genes and polypeptidefactors which may have a therapeutic potential such as induction ofregenerative processes. Additional pharmaceutical applications mayinclude the creation of new assays for toxicology and drug discovery,such as high-throughput screens for neuroprotective compounds.Generation of neural progenitors from ES cells in vitro may serve as anunlimited source of cells for tissue reconstruction and for the deliveryand expression of genes in the nervous system.

[0024] It is an object of the invention to overcome or at leastalleviate some of the problems of the prior art.

SUMMARY OF THE INVENTION

[0025] In one aspect of the present invention, there is provided anenriched preparation of undifferentiated human embryonic stem cellscapable of proliferation in vitro and differentiation to neuralprogenitor cells, neuron cells and/or glial cells.

[0026] Preferably the undifferentiated ES cells have the potential todifferentiate into neural progenitor cells, neuron cells and/or glialcells when subjected to differentiating conditions.

[0027] More preferably, the undifferentiated ES cells are capable ofmaintaining an undifferentiated state when cultured on a fibroblastfeeder layer.

[0028] In another aspect of the present invention there is provided anundifferentiated human embryonic stem cell wherein the cell isimmunoreactive with markers for human pluripotent stem cells includingSSEA-4, GCTM-2 antigen, TRA 1-60 and wherein said cell may differentiateunder differentiating conditions to neural cells. Preferably, the cellsexpress the transcription factor Oct-4 as demonstrated by RT-PCR. Morepreferably, the cells maintain a diploid karyotype during prolongedcultivation in vitro.

[0029] In another aspect there is provided an undifferentiated cell linecapable of differentiation into neural progenitor cells, neurone cellsand glial cells and preferably produced by a method of the presentinvention.

[0030] In another aspect there is provided a differentiated committedprogenitor cell line that may be cultivated for prolonged periods andgive rise to large quantities of progenitor cells.

[0031] In another aspect there is provided a differentiated committedprogenitor cell line capable of differentiation into mature neuronsand/or glial cells.

[0032] In another aspect, there is provided a neural progenitor cell,neuron cell and/or a glial cell differentiated in vitro from anundifferentiated embryonic stem cell. There is also provided a committedneural progenitor cell capable of giving rise to mature neuron cells andglial cells.

[0033] In another aspect there is provided a differentiated committedprogenitor cell line capable of establishing a graft in a recipientbrain, to participate in histogenesis of the nervous system and toconstitute the neuronal, astrocyte and oligodendrocyte lineages in vivo.

[0034] Preferably, the undifferentiated cell line is preserved bypreservation methods such as cryopreservation. Preferably the method ofcryopreservation is a method highly efficient for use with embryos suchas vitrification. Most preferably, the method includes the Open PulledStraw (OPS) vitrification method.

[0035] In another aspect the neural progenitor cell line is preserved bypreservation methods such as cryopreservation.

[0036] In another aspect, there is provided a neural progenitor cellcapable of differentiating into glial cells, including astrocytes andoligodendrocytes.

[0037] In another aspect, there is provided a neural progenitor cellcapable of transdifferentiation into other cell lineages, to generatestem cells and differentiated cells of non-neuronal phenotype, such ashemangioblast, hematopoietic stem cells, endothelial stem cells,embryonic endoderm and ectodermal cells.

[0038] In a further aspect of the present invention, there is provided amethod of preparing undifferentiated human embryonic stem cells fordifferentiation into neural progenitor cells, said method including:

[0039] obtaining an in vitro fertilised human embryo and growing theembryo to a blastocyst stage of development;

[0040] removing inner cells mass (ICM) cells from the embryo;

[0041] culturing ICM cells under conditions which do not induceextraembryonic differentiation and cell death and promote proliferationof undifferentiated cells; and

[0042] recovering the stem cells.

[0043] In a further preferred embodiment of the present invention thereis provided a method of preparing undifferentiated human embryonic stemcells for differentiation into neural progenitor cells, said methodincluding:

[0044] obtaining an in vitro fertilised human embryo;

[0045] removing inner cell mass (ICM) cells from the embryo;

[0046] culturing ICM cells on a fibroblast feeder layer to promoteproliferation of embryonic stem cells; and

[0047] recovering stem cells from the feeder layer.

[0048] In a further embodiment of the invention, the method furtherincludes:

[0049] replacing the stem cells from the fibroblast feeder layer ontoanother fibroblast feeder layer; and

[0050] culturing the stem cells for a period sufficient to promoteproliferation of morphologically undifferentiated stem cells.

[0051] In another aspect of the invention the method further includespropagating the undifferentiated stem cells.

[0052] In another aspect of the invention there is provided a method ofinducing somatic differentiation of stem cells in vitro into progenitorcells said method comprising:

[0053] obtaining undifferentiated stem cells; and

[0054] providing a differentiating signal under conditions which arenon-permissive for stem cell renewal, do not kill cells and inducesunidirectional differentiation toward extraembryonic lineages.

[0055] In a preferred embodiment of the present invention, there isprovided a method of inducing somatic differentiation of stem cells invitro into progenitor cells, said method comprising:

[0056] obtaining undifferentiated stem cells; and

[0057] culturing said cells for a prolonged period and at high densityon a fibroblast feeder cell layer to induce differentiation.

[0058] In another preferred embodiment of the present invention, thereis provided a method of inducing somatic differentiation of stem cellsin vitro into progenitor cells, said method comprising:

[0059] obtaining undifferentiated stem cells; and

[0060] transferring said cells into serum free media to inducedifferentiation.

[0061] In an additional aspect of the invention method may be used fordirecting stem cells to differentiate toward a somatic lineage.Furthermore, the method allows the establishment of a pure preparationof progenitor cells from a desired lineage and facilitate theestablishment of a pure somatic progenitor cell line.

[0062] In another preferred embodiment of the present invention, thereis provided a method of inducing the differentiation of ES derivedneural progenitor cells into differentiated mature neuronal cells, andglial cells including oligodendrocyte and astrocyte cells.

[0063] This invention provides a method that generates an in vitro andin vivo model of controlled differentiation of ES cells towards theneural lineage. The model, and the cells that are generated along thepathway of neural differentiation may be used for the study of thecellular and molecular biology of human neural development, for thediscovery of genes, growth factors, and differentiation factors thatplay a role in neural differentiation and regeneration, for drugdiscovery and for the development of screening assays for teratogenic,toxic and neuroprotective effects.

[0064] In a further aspect of the invention there is provided a neuralprogenitor cell, a neuronal cell and a glial cell that may be used forcell therapy and gene therapy.

FIGURES

[0065]FIG. 1 shows phase contrast micrographs of ES cells and theirdifferentiated progeny. A, inner cell mass three days after plating. B,colony of ES cells. C, higher magnification of an area of an ES cellcolony. D, an area of an ES cell colony undergoing spontaneousdifferentiation during routine passage. E, a colony four days afterplating in the absence of a feeder cell layer but in the presence of2000 units/ml human LIF undergoing differentiation in its periphery,. F,neuronal cells in a high density culture. Scale bars: A and C, 25microns; B and E, 100 microns; D and F, 50 microns.

[0066]FIG. 2 shows marker expression in ES cells and theirdifferentiated somatic progeny. A, ES cell colony showing histochemicalstaining for alkaline phosphatase. B. ES cell colony stained withantibody MC-813-70 recognising the SSEA-4 epitope. C, ES cell colonystained with antibody TRAL -60. D, ES cell colony stained with antibodyGCTM-2. E, high density culture, cell body and processes of a cellstained with anti-neurofilament 68 kDa protein. F, high density culture,cluster of cells and network of processes emanating from them stainedwith antibody against neural cell adhesion molecule. G, high densityculture, cells showing cytoplasmic filaments stained with antibody tomuscle actin. H, high density culture, cell showing cytoplasmicfilaments stained with antibody to desmin. Scale bars: A, 100 microns;B-D, and F, 200 microns; E, G and H, 50 microns.

[0067]FIG. 3 shows RT-PCR analysis of gene expression in ES cells andtheir differentiated derivatives. All panels show 1.5% agarose gelsstained with ethidium bromide. A, expression of Oct-4 and b-actin in ESstem cells and high density cultures. Lane 1, 100 bpDNA ladder. Lane 2,stem cell culture, b-actin. Lane 3, stem cell culture, Oct-4. Lane 4,stem cell culture, PCR for Oct-4 carried out with omission of reversetranscriptase. Lane 5, high density culture, b-actin. Lane 6, highdensity culture, Oct-4. Lane 7, high density culture, PCR for Oct-4carried out with omission of reverse transcriptase. b-actin band is 200bp and Oct-4 band is 320 bp. B, expression of nestin and Pax-6 in neuralprogenitor cells that were derived from differentiating ES colonies.Left lane, 100 bp DNA ladder; lane 1, b-actin in HX 142 neuroblastomacell line (positive control for nestin PCR); lane 2, b-actin in neuralprogenitor cells; lane 3, nestin in HX 142 neuroblastoma cell line; lane4, nestin in neural progenitor cells; lane 5, nestin PCR on same sampleas lane 4 without addition of reverse transcriptase; lane 6, Pax-6; lane7, Pax-6 PCR on same sample as line 6 without addition of reversetranscriptase. Nestin band is 208 bp, Pax-6 is 274 bp. C, expression ofglutamic acid decarboxylase in cultures of neurons. Left lane, 100 bpDNA ladder; lane 1, b-actin; lane 2, b-actin PCR on same sample as lane1 without addition of reverse transcriptase; lane 3, glutamic aciddecarboxylase; lane 4 glutamic acid decarboxylase on same sample as lane3 without addition of reverse transcriptase. Glutamic acid decarboxylaseband is 284 bp. D, expression of GABA Aα2 receptor. Left lane, 100 bpDNA ladder; lane 1, b-actin; lane 2, GABA Aα2 receptor; lane 3, PCRwithout addition of reverse transcriptase. GABA Aα2 receptor subunitband is 471 bp.

[0068]FIG. 4 shows histology of differentiated elements found interatomas formed in the testis of SCID mice following inoculation ofHES-1 or HES-2 colonies. A, cartilage and squamous epithelium, HES-2. B,neural rosettes, HES-2. C, ganglion, gland and striated muscle, HES-1.D, bone and cartilage, HES-1. E, glandular epithelium, HES-1. F,ciliated columnar epithelium, HES-1. Scale bars: A-E, 100 microns; F, 50microns.

[0069]FIG. 5 shows phase contrast microscopy and immunochemical analysisof marker expression in neural progenitor cells isolated fromdifferentiating ES cultures. A, phase contrast image of a sphere formedin serum-free medium. B-D, indirect immunofluorescence staining ofspheres, 4 hours after plating on adhesive substrate, for N-CAM, nestin,and vimentin respectively. In C and D, cells at the base of the spherewere placed in plane of focus to illustrate filamentous staining;confocal examination revealed that cells throughout the sphere weredecorated by both antibodies. Scale bar is 100 microns in all panels.

[0070]FIG. 6 shows phase contrast appearance and marker expression incultures of neurons derived from progenitor cells shown in FIG. 5. A,phase contrast micrograph of differentiated cells emanating from asphere plated onto adhesive surface. B-H, indirect immunofluorescencemicroscopy of differentiated cells decorated with antibodies against 200kDa neruofilament protein (B), 160 kDa neurofilament protein (C),MAP2a+b (D), glutamate (E), synaptophysin (F), glutamic aciddecarboxylase (G) and β-tubulin (H). Scale bars: A, ;B, 100 microns; C,200mircons; D, 20 microns; E and F, 10 microns; G, 20 microns; H, 25microns.

[0071]FIG. 7 shows neural precursors proliferating as a monolayer on aplastic tissue culture dish in the presence of EGF and bFGF. Thesemonolayer cultures of proliferating cells were obtained after prolongedcultivation (2-3 weeks) of the spheres in the presence of growth factorswithout sub-culturing.

[0072]FIG. 8 shows phase contrast appearance of a culture consisting ofdifferentiated neural cells.

[0073]FIG. 9 shows phase contrast appearance of a sphere that is formed72 hours after the transfer of a clump of undifferentiated ES cells intoserum free medium (Scale bar 100 microns).

[0074]FIG. 10 shows linear correlation between the volume of spheres andthe number of progenitor cells within a sphere. Spheres of variousdiameters that were generated from differentiating ES colonies and werepropagated for 14-15 weeks were dissaggregated into single cellsuspension and the number of cells per sphere was counted.

[0075]FIG. 11 shows indirect immunofluorescence staining of a sphere, 4hours after plating on adhesive substrate, for N-CAM. The sphere wasgenerated by direct transfer of undifferentiated ES cells into serumfree medium and propagation of the resulting spheres for 5 passages.(Scale bar 100 microns).

[0076]FIG. 12 shows indirect immunofluorescence membraneous staining forN-CAM of single cells at the periphery of a sphere 4 hours after platingon adhesive substrate. The sphere was generated by direct transfer ofundifferentiated ES cells into serum free medium and propagation of theresulting spheres for 5 passages. (Scale bar 25 microns).

[0077]FIG. 13 shows indirect immunofluorescence staining of a spheres 4hours after plating on adhesive substrate for the intermediate filamentnestin. Cells at the base of the sphere were placed in plane of focus toillustrate filamentous staining. The sphere was generated by directtransfer of undifferentiated ES cells into serum free medium andpropagation of resulting spheres for 5 passages. (Scale bar 25 microns).

[0078]FIG. 14 shows indirect immunofluorescence microscopy of adifferentiated cell decorated with antibodies against theoligodendrocyte progenitor marker O4. (Scale bar 12.5 microns).

[0079]FIG. 15 shows indirect immunofluorescence staining of a sphere 4hours after plating on adhesive substrate for the intermediate filamentvimentin. Cells at the base of the sphere were placed in plane of focusto illustrate filamentous staining. The sphere was generated by directtransfer of undifferentiated ES cells into serum free medium andpropagation of resulting spheres for 7 passages. (Scale bar 25 microns).

[0080]FIG. 16 shows the growth pattern of spheres that were generateddirectly from undifferentiated ES cells. Each bar represents the mean(±SD) increment in volume per week of 24 spheres at first to sixteenweeks after derivation. A more excessive growth rate is evident duringthe first 5 weeks.

[0081]FIG. 17 shows persistent growth in the volume of spheres alongtime. Each bar represents the mean (±SD) increment in volume per week of24 spheres at nine to twenty one weeks after derivation. The sphereswere generated from differentiating ES colonies.

[0082]FIG. 18 shows linear correlation between the volume of spheres andthe number of progenitor cells within a sphere. Spheres of variousdiameters, that were generated directly from undifferentiated ES cellsand were propagated 5-7 weeks, were dissaggregated into single cellsuspension and the number of cells per sphere was counted.

[0083]FIG. 19 shows RT-PCR analysis of gene expression in ES cells (aweek after passage) and neural spheres derived from differentiatingcolonies and directly from undifferentiated ES cell. All panels show 2%agarose gels stained with ethidium bromide. Lanes 1, 2 and 3, Oct-4 inES cell culture, neural spheres derived from differentiating colonies,neural spheres derived from undifferentiated ES cells. Lane 4, stem cellculture, PCR for Oct-4 carried out with omission of reversetranscriptase. Lanes 5, 6, and 7, nestin in ES cell culture, neuralspheres derived from differentiating colonies, neural spheres derivedfrom undifferentiated ES cells. Lane 8, stem cell culture, PCR fornestin carried out with omission of reverse transcriptase. Lanes 9, 10and 11, Pax-6 in ES cell culture, neural spheres derived fromdifferentiating colonies, neural spheres derived from undifferentiatedES cells. Lane 12, stem cell culture, PCR for Pax-6 carried out withomission of reverse transcriptase. Lane 13, 100 bp DNA ladder. Oct-4band is 320 bp, nestin is 208 bp and Pax-6 is 274 bp.

[0084]FIG. 20 shows indirect immunofluorescence microscopy ofdifferentiated astrocyte cells decorated with antibody against GFAP.(Scale bar 25 microns).

[0085]FIG. 21 shows indirect immunofluorescence microscopy of brainsections of two mice (A and B) 4 weeks after transplantation of humanneural precursors prelabeled with BrDU. Cells with a nucleus decoratedwith anti BrDU (brown stain, black arrow) are evident near theventricular surface (white arrow indicate mouse unstained nuclei, bar=20microns).

[0086]FIG. 22 shows indirect immunofluorescence microscopy of brainsections of a mice 4 weeks after transplantation of human neuralprecursors prelabeled with BrDU. Wide spread distribution oftransplanted human cells decorated by anti BrDU antibodies is evident inthe periventricular areas. The periventricular area in A is demonstratedat a higher magnification in B and C. (Bars=150, 60 and 30 microns in A,B and C).

[0087]FIG. 23 shows indirect immunocytochemical microscopy of brainsections of a mice 4 weeks after transplantation of human neuralprecursors prelabeled with BrDU. The transplanted human cells aremigrating along the rostral migratory stream (bar=150 microns).

[0088]FIG. 24 shows RT-PCR analysis of gene expression in neural spheresderived from differentiating (A) and undifferentiated (B) ES cells. Allpanels show 2% agarose gels stained with ethidium bromide. Lanes 1 and10, 100 bpDNA ladder; Lane 2, CD-34; Lane 3, Flk-1; lane4, HNF-3; lane5, alfafetoprotein. Lanes 6-9 PCR reaction on the same samples as lanes2-5 carried out with the omission of reverse transcriptase. CD-34 bandis 200 bp, Flk-1 is 199, HNF-3 is 390, AFP is 340 bp.

[0089]FIG. 25 shows by RT-PCR analysis the expression of GFAP and thepip gene in differentiated cells from neural spheres derived fromdifferentiating ES cell colonies. The expression of GFAP indicatesdifferentiation into astrocytes while the presence of both dm-20 and piptranscripts indicate that differentiation into oligodendrocyte cells hasoccurred. Lanes 2, 4, 6 and lanes 3, 5, 7 are from two separate RNAsamples from differentiated spheres that were independently derived fromES cells. Lane 1 and 8, 100 bp DNA ladder; Lanes 2 and 4, GFAP; lanes 3and 5, plp and dm-20; lanes 6 and 7, PCR reaction on the same samples aslanes 3 and 5 carried out with the omission of reverse transcriptase.GFAP band is 383, pip band is 354 bp and dm-20 is 249 bp.

[0090]FIG. 26 shows a dark field stereomicroscopic photograph of areas(arrows) destined to give rise to neural precursors in a differentiatingES cell colony 3 weeks after passage (bar=1.6 mm).

[0091]FIG. 27 shows indirect immunochemical analysis of markerexpression in cultures of neurons derived from progenitor cells thatwere derived directly from undifferentiated ES cells: A, indirectimmunofluorescence microscopy of neurits decorated with antibody against160 kDa neurofilament protein. B and C, indirect immunofluorescencestaining of differentiated cells for MAP2a+b and β-tubulin III. Scalebars: A 100 microns, B and C 10 microns.

[0092]FIG. 28 shows indirect immunochemical analysis of the expressionof tyrosine hydroxylase. Neurits (A) and a differentiated cell (B) aredecorated with antibodies against tyrosine hydroxylase. Scale bars: 30microns.

[0093]FIG. 29 shows in vivo differentiation into astrocyte cells oftransplanted human neural progenitors prelabeled with BrDU. Donor cellsare identified by indirect immunochemical detection of BrDU (darknuclei, arrows). Duel staining demonstrates donor cells decorated byanti GFAP (orange). Transplanted cells are migrating into the brainparenchyma (white arrow) and are also found in the periventricular zone(dark arrow) (A), A higher magnification of cells that havedifferentiated into astrocytes and migrated into the host brain (B).

[0094]FIG. 30 shows in vivo differentiation into oligodendrocyte cellsof transplanted human neural progenitors prelabeled with BrDU. Donorcells are identified by indirect immunochemical detection of BrDU (darknuclei, arrows). Duel staining demonstrates donor cells decorated byanti CNPase (orange).

[0095]FIG. 31 shows cumulative growth curve for human neural progenitorsderived from differentiating colonies. (A) Continuous growth is evidentduring an 18-22 week period. The increment in the volume of the sphereswas continuously monitored as an indirect measure of the increase incell numbers. A linear positive correlation between the volume of thespheres and the number of cells within the spheres (B, insert) wasmaintained along cultivation. It supported the validity of monitoringthe increment of sphere volume as an indirect indicator of cellproliferation.

[0096]FIG. 32 shows RT-PCR analysis of the expression of non-neuralmarkers in human ES derived spheres. All panels show 2% agarose gelsstained with ethidium bromide. The symbols + and − indicate whether thePCR reaction was performed with or without the addition of reversetranscriptase. A 1 Kb plus DNA ladder was used in all panels. β-actinband is 291 bp, keratin is 780 bp, Flk-1 is 199 bp, CD34 is 200 bp,AC-133 is 200 bp, transferin is 367 bp, amylase is 490 bp and α1 antitrypsin is 360 bp.

[0097]FIG. 33 shows a phase contrast micrograph of differentiated cellsgrowing out from a sphere 2 weeks after plating onto an adhesive surfaceand culture in the absence of growth factors. Scale bar is 200 μm.

[0098]FIG. 34 shows RT-PCR analysis of the expression of neuronal andglial markers in differentiated cells originating from human ES derivedneural spheres. All panels show 2% agarose gels stained with ethidiumbromide. The symbols + and − indicate whether the PCR reaction wasperformed with or without the addition of reverse transcriptase. A 1 Kbplus DNA ladder was used in all panels. Plp and dm-20 bands are 354 bpand 249 bp respectively, MBP is 379 bp, GFAP is 383 bp, NSE is 254 bpand NF-M is 430 bp.

[0099]FIG. 35 shows indirect immunochemical analysis of the expressionof serotonin (A) and GABA (B). Scale bars are 20 μm.

[0100]FIG. 36 shows dissemination of transplanted BrdU+ human ES-derivedneural progenitor cells in the mouse host brain.

[0101] (A) At 2 days after transplantation most cells were found liningthe ventricular wall. (B) After 4-6 weeks most cells had left theventricles (V) and populated the corpus callosum (CC), fimbria (fim),internal capsule (i.c.). BrdU+ cells were not found in the striatum(str) or CA region of the hippocampus (hipp). (C) Chains of BrdU+ cellswere found in the rostral migratory stream (RMS). (D) BrdU+ cells in theperiventricular white matter. (E) Higher magnification of D, to shownuclear specific localization of BrdU.

[0102]FIG. 37 shows identification of the transplanted cells in thebrain by human and neural-lineage specific markers. (A) A typical chainof transplanted cells in the corpus callosum, stained with humanspecific anti-mitochondrial antibody. The mitochondrial staining (greenfluorescence) on Nomarsky background (blue, cell nuclei indicated byasterisk) shows a typical perinuclear localization. (B) Double stainingfor BrdU (green fluorescence) and human specific anti ribonuclearprotein (red fluorescence) shows nuclear co-localization, indicatingthat BrdU+ cells were indeed of human origin. (C) A GFAP+ astrocyte(red) from the periventricular region, co-labeled with BrdU (green),indicating its origin from the graft. (D) An NG2+ oligodendrocyteprogenitor (red) in the periventricular region, co-labeled with BrdU(green). (E) A CNPase+ oligodendrocyte (red) in the corpus callosum,co-labeled with BrdU (immunohistochemistry, shown as dark nucleus inNomarsky). (F) Neuronal processes in the fimbria, stained with a humanspecific anti- 70 kDa neurofilament. (G) A βIII-tubulin+ neuron (greenfluorescence) in the olfactory bulb, co-labeled with BrdU (as darknucleus (arrow) in Nomarsky). Bars=10 μm.

DESCRIPTION OF THE INVENTION

[0103] In one aspect of the present invention there is provided anenriched preparation of human undifferentiated embryonic stem cellscapable of proliferation in vitro and differentiation to neuralprogenitor cells, neuron cells and/or glial cells.

[0104] Throughout the description and claims of this specification, theword “comprise” and variations of the word, such as “comprising” and“comprises”, is not intended to exclude other additives, components,integers or steps. Established pluripotent ES cell lines from humanblastocysts are shown in, PCT/AU99/00990 by the applicants. In contrastto data that has published previously, the human ES cell lines that havebeen derived by the applicants in the present application have beenshown to differentiate in vitro into somatic lineages and give rise toneurons and muscle cells. Moreover, Applicants have demonstrated thederivation of neural progenitor cells from human ES cells in vitro.These ES derived human neural progenitors may give rise to matureneurons in vitro. The contents of PCT/AU99/00990 are herebyincorporated.

[0105] Proliferation in vitro may include cultivation of the cells forprolonged periods. The cells are substantially maintained in anundifferentiated state. Preferably the cells are maintained underconditions which do not induce cell death or extraembryonicdifferentiation.

[0106] Preferably, they are capable of maintaining an undifferentiatedstate when cultured on a fibroblast feeder layer preferably undernon-differentiating conditions. Preferably the fibroblast feeder layerdoes not induce extraembryonic differentiation.

[0107] More preferably the cells have the potential to differentiate invitro when subjected to differentiating conditions. Most preferably thecells have the capacity to differentiate in vitro into a wide array ofsomatic lineages.

[0108] The promotion of stem cells capable of being maintained in anundifferentiated state in vitro on one hand, and which are capable ofdifferentiation in vitro into extraembryonic and somatic lineages on theother hand, allows for the study of the cellular and molecular biologyof early human development, functional genomics, generation ofdifferentiated cells from the stem cells for use in transplantation ordrug screening and drug discovery in vitro.

[0109] Once the cells are maintained in the undifferentiated state, theymay be differentiated to mature functional cells. The embryonic stemcells are derived from the embryo and are pluripotent and have thecapability of developing into any organ or tissue type. Preferably thetissue type is selected from the group including endocrine cells, bloodcells, neural cells or muscle cells. Most preferably they are neuralcells.

[0110] In another aspect of the present invention there is provided anundifferentiated human embryonic stem cell wherein the cell isimmunoreactive with markers for human pluripotent stem cells includingSSEA-4, GCTM-2 antigen, and TRA 1-60 and wherein said cell candifferentiate, under differentiating conditions to neural cells.Preferably, the cells express specific transcription factors such asOct-4 as demonstrated by RT-PCR, or methods of analysis of differentialgene expression, microarray analysis or related techniques. Morepreferably the cells maintain a diploid karyotype during prolongedcultivation in vitro. Preferably, the stem cell will constitute anenriched preparation of an undifferentiated stem cell line. Morepreferably, the stem cell line is a permanent cell line, distinguishedby the characteristics identified above. They preferably have normalkaryotype along with the characteristics identified above. Thiscombination of defining properties will identify the cell lines of theinvention regardless of the method used for their isolation.

[0111] Methods of identifying these characteristics may be by any methodknown to the skilled addressee. Methods such as (but not limited to)indirect immunoflourescence or immunocytochemical staining may becarried out on colonies of ES cells which are fixed by conventionalfixation protocols then stained using antibodies against stem cellspecific antibodies and visualised using secondary antibodies conjugatedto fluorescent dyes or enzymes which can produce insoluble coloredproducts. Alternatively, RNA may be isolated from the stem cells andRT-PCR or Northern blot analysis carried out to determine expression ofstem cell specific genes such as Oct-4.

[0112] In a preferred embodiment the undifferentiated cells form tumourswhen injected in the testis of immunodeprived SCID mice. These tumoursinclude differentiated cells representative of all three germ layers.The germ layers are preferably endoderm, mesoderm and ectoderm.Preferably, once the tumours are established, they may be disassociatedand specific differentiated cell types may be identified or selected byany methods available to the skilled addressee. For instance, lineagespecific markers may be used through the use of fluorescent activatedcell sorting (FACS) or other sorting method or by direct microdissection of tissues of interest. These differentiated cells may beused in any manner. They may be cultivated in vitro to produce largenumbers of differentiated cells that could be used for transplantationor for use in drug screening for example.

[0113] In another aspect there is provided a differentiated committedprogenitor cell line capable of differentiation and propagation intomature neurons and/or glial cells. The undifferentiated cells maydifferentiate in vitro to form neural progenitor cells, neuron cellsand/or glial cells.

[0114] In another aspect, there is provided a neural progenitor cell,neuron cell and/or glial cell differentiated in vitro from anundifferentiated embryonic stem cell. There is also provided a committedneural progenitor cell capable of giving rise to mature neuron cells.The mature neuron cell may be characterized by an ability to express 16kDa neuro-ilament protein, MAP2ab, glutamate, synaptophysin, glutamicacid decarboxylase (GAD), tyrosine hydroxylase, GABA and serotonin.

[0115] In another aspect, there is provided a neural progenitor cellcapable of differentiating into glial cells, including astrocytes andoligodendrocytes. The glial cells include microglial cells and radialglial cells.

[0116] In another aspect, there is provided a neural progenitor cellcapable of transdifferentiation into other cell lineages, to generatestem cells and differentiated cells of non-neuronal phenotype, such ashemangioblast hematopoietic stem cells or endothelial stem cells,embryonic endoderm cells and ectoderm cells.

[0117] These cells may be obtained by somatic differentiation of humanES cells, identified by neural markers. These cells may be isolated inpure form from differentiating ES cells, in vitro, and propagated invitro. They may be induced to under go differentiation to mature neuronsand/or glial cells.

[0118] The cells may undergo differentiation in vitro to yield neuralprogenitor cells, neuron or glial cells as well as extraembryonic cells,such differentiation being characterised by novel gene expressioncharacteristic of specific lineages as demonstrated byimmunocytochemical or RNA analysis. Characterisation may be obtained byusing expression of genes characteristic of pluripotent cells orparticular lineages. Preferably, differential expression of Oct-4 may beused to identify stem cells from differentiated cells. Otherwise, thepresence or absence of expression of other genes characteristic ofpluripotent stem cells or other lineages may include Genesis, GDF-3 orCripto. Analysis of these gene expressions may create a gene expressionprofile to define the molecular phenotype of an ES cell, a committedprogenitor cell, or a mature differentiated cell of any type. Suchanalysis of specific gene expression in defined populations of cellsfrom ES cultures is called cytomics. Methods of analysis of geneexpression profiles include RT-PCR, methods of differential geneexpression, microarray analysis or related techniques.

[0119] Differentiating cultures of the stem cells secrete humanchorionic gonadotrophin (hCG) and α-fetoprotein (AFP) into culturemedium, as determined by enzyme-linked immunosorbent assay carried outon culture supernatants. Hence this may also serve as a means ofidentifying the differentiated cells.

[0120] The differentiated cells forming neural progenitor cells, neuroncells and/or glial cells may also be characterised by expressed markerscharacteristic of differentiating cells. The in vitro differentiatedcell culture may be identified by detecting molecules such as markers ofthe neuroectodermal lineage, markers of neural progenitor cells,neuro-filament proteins, monoclonal antibodies such as MAP2ab,glutamate, synaptophysin, glutamic acid decarboxylase, GABA, serotonin,tyrosine hydroxylase, β-tubulin, β-tubulin III, GABA Aα2 receptor, glialfibrillary acidic protein (GFAP), , 2′, 3′-cyclic nucleotide3′-phosphodiesterase (CNPase), plp, DM-20, O4, and NG-2 staining.

[0121] In another preferred aspect of the present invention there isprovided a neural progenitor cell wherein the cell express markers forthe neuroectodermal lineage as well as neural markers selected from thegroup including polysialyated N-CAM, N-CAM, A2B5, nestin, vimentin andthe transcriptional factor Pax-6, and do not express Oct-4.

[0122] Preferably, the cells do not express the transcriptional factorOCT-4. This may be demonstrated by RT-PCR, or methods of analysis ofdifferential gene expression, microarray analysis or related techniques.More preferably the cells will constitute an enriched preparation. Theycan proliferate in vitro for prolonged periods at an undifferentiatedneural progenitor state to produce a large number of cells. The neuralprogenitor cells can differentiate, under differentiating conditions tomature neurons and glial cells.

[0123] In yet another aspect, the invention provides a neural progenitorcell which is capable of establishing a graft in a recipient brain.Preferably the neural progenitor cell is as described above.

[0124] Upon transplantation to the developing brain they incorporateextensively into the host brain, demonstrate wide spread distribution,migrate along established host brain migratory tracks, differentiate ina region specific manner into progeny of the three fundamental neurallineages, indicating their capability to respond to local cues andparticipate in the development and histogenesis of the living host. Thiscombination of defining properties will identify the neural progenitorcell lines of the invention regardless of the method used for theirisolation.

[0125] In yet another aspect of the present invention, there is provideda glial cell differentiated from a neural progenitor cell. Preferably,the glial cell is an astrocyte or an oligodendrocyte. Oligodendrocytesmay be identified by O4 and NG-2 immunostaining or by RNA transcripts ofmyelin basic protein (MBP), plp and dm-20.

[0126] In a further aspect of the invention, there is provided a methodof preparing undifferentiated human embryonic stem cells fordifferentiation into neural progenitor cells, said method including:

[0127] obtaining an in vitro fertilised human embryo and growing theembryo to a blastocyst stage of development;

[0128] removing inner cells mass (ICM) cells from the embryo;

[0129] culturing ICM cells under conditions which do not induceextraembryonic differentiation and cell death, and promote proliferationof undifferentiated stem cells; and

[0130] recovering the stem cells.

[0131] The stem cells will be undifferentiated cells and can be inducedto differentiate when a differentiating signal is applied.

[0132] In a preferred embodiment of the present invention there isprovided a method of preparing undifferentiated human embryonic stemcells for differentiation into neural progenitor cells, said methodincluding:

[0133] obtaining an in vitro fertilised human embryo;

[0134] removing inner cells mass (ICM) cells from the embryo;

[0135] culturing ICM cells on a fibroblast feeder layer to promoteproliferation of embryonic stem cells; and

[0136] recovering stem cells from the feeder layer.

[0137] Embryonic stem cells (ES) are derived from the embryo. Thesecells are undifferentiated and have the capability of differentiation toa variety of cell types. The “embryo” is defined as any stage afterfertilization up to 8 weeks post conception. It develops from repeateddivision of cells and includes the stages of a blastocyst stage whichcomprises an outer trophectoderm and an inner cell mass (ICM).

[0138] The embryo required in the present method may be an in vitrofertilised embryo or it may be an embryo derived by transfer of asomatic cell or cell nucleus into an enucleated oocyte of human or nonhuman origin which is then activated and allowed to develop to theblastocyst stage.

[0139] The embryo may be fertilised by any in vitro methods available.For instance, the embryo may be fertilised by using conventionalinsemination, or intracytoplasmic sperm injection.

[0140] An embryo that is recovered from cryopreservation is alsosuitable. An embryo that has been cryopreserved at any stage ofdevelopment is suitable. Preferably embryos that were cryopreserved atthe zygote or cleavage stage are used. Any method of cryopreservation ofembryos may be used. It is preferred that a method producing highquality (good morphological grade) embryos is employed.

[0141] It is preferred that any embryo culture method is employed but itis most preferred that a method producing high quality (goodmorphological grade) blastocysts is employed. The high quality of theembryo can be assessed by morphological criteria. Most preferably theinner cell mass is well developed. These criteria can be assessed by theskilled addressee.

[0142] Following insemination, embryos may be cultured to the blastocyststage. Embryo quality at this stage may be assessed to determinesuitable embryos for deriving ICM cells. The embryos may be cultured inany medium that maintains their survival and enhances blastocystdevelopment.

[0143] Preferably, the embryos are cultured in droplets underpre-equilibrated sterile mineral oil in IVF-50 or Scandinavian 1 (S1) orG2 medium (Scandinavian IVF). Preferably the incubation is for two days.If IVF-50 or S1 is used, on the third day, an appropriate medium such asa mixture of 1:1 of IVF-50 and Scandinavian-2 medium (Scandinavian IVF)may be used. From at least the fourth day, a suitable medium such asG2.2 or Scandinavian-2 (S2) medium may be used solely to grow theembryos to blastocyst stage (blastocysts). Preferably, only G2.2 mediumis used from the fourth day onwards.

[0144] In a preferred embodiment, the blastocyst is subjected toenzymatic digestion to remove the zona pellucida or a portion thereof.Preferably the blastocyst is subjected to the digestion at an expandedblastocyst stage which may be approximately on day 6. Generally this isat approximately six days after insemination.

[0145] Any protein enzyme may be used to digest the zona pellucida orportion thereof from the blastocyst. Examples include pronase, acidTyrodes solution, and mechanical methods such as laser dissection.

[0146] Preferably, Pronase is used. The pronase may be dissolved in PBSand G2 or S2 medium. Preferably the PBS and Scandinavian-2 medium isdiluted 1:1. For digestion of zona pellucida from the blastocyst,approximately 10 units/ml of Pronase may be used for a period sufficientto remove the zona pellucida. Preferably approximately 1 to 2 mins, morepreferably 1 to 1.5 mins is used.

[0147] The embryo (expanded blastocyst) may be washed in G2.2 or S2medium, and further incubated to dissolve the zona pellucida.Preferably, further digestion steps may be used to completely dissolvethe zona. More preferably the embryos are further incubated in pronasesolution for 15 seconds. Removal of the zona pellucida thereby exposesthe trophectoderm.

[0148] In a preferred embodiment of the invention the method furtherincludes the following steps to obtain the inner cell mass cell, saidsteps including:

[0149] treating the embryo to dislodge the trophectoderm of the embryoor a portion thereof;

[0150] washing the embryo with a G2.2 or S2 medium to dislodge thetrophectoderm or a portion thereof; and

[0151] obtaining inner cell mass cells of the embryo.

[0152] Having had removed the zona pellucida, the ICM and trophectodermbecome accessible. Preferably the trophectoderm is separated from theICM. Any method may be employed to separate the trophectoderm from theICM. Preferably the embryo (or blastocyst devoid of zona pellucida) issubjected to immuno-surgery. Preferably it is treated with an antibodyor antiserum reactive with epitopes on the surface of the trophectoderm.More preferably, the treatment of the embryo, (preferably an embryo atthe blastocyst stage devoid of zona pellucida) is combined withtreatment with complement. The antibody and/or antiserum and complementtreatment may be used separately or together. Preferred combinations ofantibody and/or antiserum and complement include anti-placental alkalinephosphatase antibody and Baby Rabbit complement (Serotec) or anti-humanserum antibody (Sigma) combined with Guinea Pig complement (Gibco).

[0153] Preferably the antibodies and complement are diluted in G2.2 orS2 medium. The antibodies and complement, excluding anti-placentalalkaline phosphate (anti-AP) are diluted 1:5 whereas anti-AP antibody isdiluted 1:20 with S-2 medium.

[0154] Preferably the embryo or blastocyst (preferably having the zonapellucida removed) is subjected to the antibody before it is subjectedto the complement. Preferably, the embryo or blastocyst is cultured inthe antibody for a period of approximately 30 mins.

[0155] Following the antibody exposure, it is preferred that the embryois washed. Preferably it is washed in G2.2 or S2 medium. The embryo orblastocyst preferably is then subjected to complement, preferably for aperiod of approximately 30 mins.

[0156] G2.2 or S2 (Scandinavian-2) medium is preferably used to wash theembryo or blastocyst to dislodge the trophectoderm or a portion thereof.Dislodgment may be by mechanical means. Preferably the dislodgment is bypipetting the blastocyst through a small bore pipette.

[0157] The ICM cells may then be exposed and ready for removal andculturing. Culturing of the ICM cells may be conducted on a fibroblastfeeder layer. In the absence of a fibroblast feeder layer, the cellswill differentiate. Leukaemia inhibitory factor (LIF) has been shown toreplace the feeder layer in some cases and maintain the cells in anundifferentiated state. However, this seems to only work for mousecells. For human cells, high concentrations of LIF were unable tomaintain the cells in an undifferentiated state in the absence of afibroblast feeder layer.

[0158] The conditions which do not induce extraembryonic differentiationand cell death may include cultivating the embryonic stem cells on afibroblast feeder layer which does not induce extraembryonicdifferentiation and cell death.

[0159] Mouse or human fibroblasts are preferably used. They may be usedseparately or in combination. Human fibroblasts provide support for stemcells, but they create a non-even and sometimes non-stable feeder layer.

[0160] However, they may combine effectively with mouse fibroblasts toobtain an optimal stem cell growth and inhibition of differentiation.

[0161] The cell density of the fibroblast layer affects its stabilityand performance. A density of approximately 25,000 human and 70,000mouse cells per cm² is most preferred. Mouse fibroblasts alone are usedat 75,000-100,000/cm². The feeder layers are preferably established 6-48hours prior to addition of ES or ICM cells.

[0162] Preferably the mouse or human fibroblast cells are low passagenumber cells. The quality of the fibroblast cells affects their abilityto support the stem cells. Embryonic fibroblasts are preferred. Formouse cells, they may be obtained from 13.5 day old foetuses. Humanfibroblasts may be derived from embryonic or foetal tissue fromtermination of pregnancy and may be cultivated using standard protocolsof cell culture.

[0163] The guidelines for handling the mouse embryonic fibroblasts mayinclude minimising the use of trypsin digestion and avoidance ofovercrowding in the culture. Embryonic fibroblasts that are not handledaccordingly will fail to support the growth of undifferentiated EScells. Each batch of newly derived mouse embryonic fibroblasts is testedto confirm its suitability for support and maintenance of stem cells.

[0164] Fresh primary embryonic fibroblasts are preferred in supportingstem cell renewal and/or induction of somatic differentiation ascompared to frozen-thawed fibroblasts. Nevertheless, some batches willretain their supportive potential after repeated freezing and thawing.Therefore each fresh batch that has proved efficient in supporting EScells renewal and/or induction of somatic differentiation is retestedafter freezing and thawing. Batches that retain their potential afterfreezing and thawing are most preferably used. Batches are tested todetermine suitability for the support of stem cell renewal, theinduction of somatic differentiation or the induction of extraembryonicdifferentiation.

[0165] Some mouse strains yield embryonic fibroblasts which are moresuitable for stem cell maintenance and induction of somaticdifferentiation than those of other strains. For example, fibroblastsderived from inbred 129/Sv or CBA mice or mice from a cross of 129/Svwith C57/BI6 strains have proven highly suitable for stem cellmaintenance.

[0166] Isolated ICM masses may be plated and grown in culture conditionssuitable for human stem cells.

[0167] It is preferred that the feeder cells are treated to arrest theirgrowth. Several methods are available. It is preferred that they areirradiated or are treated with chemicals such as mitomycin C thatarrests their growth. Most preferably, the fibroblast feeder cells aretreated with mitomycin C (Sigma).

[0168] The fibroblast feeder layer maybe generally plated on a gelatintreated dish. Preferably, the tissue culture dish is treated with 0.1%gelatin.

[0169] The fibroblast feeder layer may also contain modifiedfibroblasts. For instance, fibroblasts expressing recombinant membranebound factors essential for stem cell renewal may be used. Such factorsmay include for example human multipotent stem cell factor.

[0170] Inner cell mass cells may be cultured on the fibroblast feederlayer and maintained in an ES medium. A suitable medium is DMEM (GIBCO,without sodium pyruvate, with glucose 4500 mg/L) supplemented with 20%FBS (Hyclone, Utah), (betamercaptoethanol-0.1 mM (GIBCO), non essentialamino acids-NEAA-1% (GIBCO), glutamine 2 mM. (GIBCO), and penicillin 50μ/ml, streptomycin 50 μg/ml (GIBCO). In the early stages of ES cellcultivation, the medium maybe supplemented with human recombinantleukemia inhibitory factor hLIF preferably at 2000 μ/ml. However, LIFgenerally is not necessary. Any medium may be used that can support theES cells.

[0171] The ES medium may be further supplemented with soluble growthfactors which promote stem cell growth or survival or inhibit stem celldifferentiation. Examples of such factors include human multipotent stemcell factor, or embryonic stem cell renewal factor.

[0172] The isolated ICM may be cultured for at least six days. At thisstage, a colony of cells develops. This colony is comprised principallyof undifferentiated stem cells. They may exist on top of differentiatedcells. Isolation of the undifferentiated cells may be achieved bychemical or mechanical means or both. Preferably mechanical isolationand removal by a micropipette is used. Mechanical isolation may becombined with a chemical or enzymatic treatment to aid with dissociationof the cells, such as Ca²⁺/Mg²⁺ free PBS medium or dispase.

[0173] In a further preferred embodiment of the invention, the methodfurther includes:

[0174] replating the stem cells from the fibroblast feeder layer ontoanother fibroblast feeder layer; and

[0175] culturing the stem cells for a period sufficient to obtainproliferation of morphologically undifferentiated stem cells.

[0176] A further replating of the undifferentiated stem cells isperformed. The isolated clumps of cells from the first fibroblast feederlayer may be replated on fresh human/mouse fibroblast feeder layer inthe same medium as described above.

[0177] Preferably, the cells are cultured for a period of 7-14 days.After this period, colonies of undifferentiated stem cells may beobserved. The stem cells may be morphologically identified preferably bythe high nuclear/cytoplasmic ratios, prominent nucleoli and compactcolony formation. The cell borders are often distinct and the coloniesare often flatter than mouse ES cells. The colonies resemble thoseformed by pluripotent human embryonal carcinoma cell lines such as GCT27 X-1.

[0178] In another embodiment of the invention, the method furtherincludes propagating the undifferentiated stem cells. The methods ofpropagation may initially involve removing clumps of undifferentiatedstem cells from colonies of cells. The dispersion is preferably bychemical or mechanical means or both. More preferably, the cells arewashed in a Ca²⁺/Mg²⁺ free PBS or they are mechanically severed from thecolonies or a combination of these methods or any known methodsavailable to the skilled addressee. In these methods, cells may bepropagated as clumps of about 100 cells about every 7 days.

[0179] In the first method, Ca²⁺/Mg²⁺ free PBS medium may be used toreduce cell-cell attachments. Following about 15-20 minutes, cellsgradually start to dissociate from the monolayer and from each other anddesired size clumps can be isolated. When cell dissociation is partial,mechanical dissociation using the sharp edge of the pipette may assistwith cutting and the isolation of the clumps.

[0180] An alternative chemical method may include the use of an enzyme.The enzyme may be used alone or in combination with a mechanical method.Preferably, the enzyme is dispase.

[0181] An alternative approach includes the combined use of mechanicalcutting of the colonies followed by isolation of the subcolonies bydispase. Cutting of the colonies may be performed in PBS containing Ca²⁺and Mg²+. The sharp edge of a micropipette may be used to cut thecolonies to clumps of about 100 cells. The pipette may be used to scrapeand remove areas of the colonies. The PBS is preferably changed toregular equilibrated human stem cell medium containing dispase (Gibco)10 mg/ml and incubated for approximately 5minutes at 37° C. in ahumidified atmosphere containing 5% CO₂. As soon as the clumps detachedthey may be picked up by a wide bore micro-pipette, washed in PBScontaining Ca²⁺ and Mg²⁺ and transferred to a fresh fibroblast feederlayer.

[0182] The fibroblast feeder layer may be as described above.

[0183] Undifferentiated embryonic stem cells have a characteristicmorphology as described above. Other means of identifying the stem cellsmay be by cell markers or by measuring expression of genescharacteristic of pluripotent cells.

[0184] Examples of genes characteristic of pluripotent cells orparticular lineages may include (but are not limited to) Oct-4 andPax-6, polysialyated N-CAM, N-CAM, A2B5, nestin and vimentin as markersof stem cells and neuronal precursors respectively. Other genescharacteristic of stem cells may include Genesis, GDF-3 and Cripto.CD-34 is characteristic of hematopoietic stem cells and flk-1 isexpressed by the hemangioblast. AC-133 may be characteristic of bothhematopoietic and neural progenitors. Keratin is characteristic ofepidermal cells while transferin, amylase and α1 anti-trypsin arecharacteristic of embryonic endoderm. Such gene expression profiles maybe attained by any method including RT-PCR, methods of differential geneexpression, microarray analysis or related techniques.

[0185] Preferably the stem cells may be identified by beingimmunoreactive with markers for human pluripotent stem cells includingSSEA-4, GCTM-2 antigen, TRA 1-60. Preferably the cells express thetranscription factor Oct-4. The cells also maintain a diploid karyotype.Preferably the neural progenitor cells are identified by expressedmarkers of primitive neuroectoderm and neural stem cells such as N-CAM,polysialyated N-CAM, A2B5, intermediate filament proteins such as nestinand vimentin and the transcription factor Pax-6. Neurons may beidentified by structural markers such as β-tubulin, β-tubulin III, the68 kDa and the 200 kDa neurofilament proteins. Mature neurons may alsobe identified by the 160 kDa neurofilament proteins, Map-2a, b andsynaptophysin, glutamate, GABA, serotonin, tyrosine hydroxylase, GABAbiosynthesis and receptor subunits characteristic of GABA minergicneurons (GABA Aα2). Astrocytes may be identified by the expression ofglial fibrillary acidic protein (GFAP), and oligodendrocyte by 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), plp, DM-20, myelinbasic protein (MBP), NG-2 staining and O4.

[0186] The stem cells may be further modified at any stage of isolation.They may be genetically modified through introduction of vectorsexpressing a selectable marker under the control of a stem cell specificpromoter such as Oct-4. Some differentiated progeny of embryonic stemcells may produce products that are inhibitory to stem cell renewal orsurvival. Therefore selection against such differentiated cells,facilitated by the introduction of a construct such as that describedabove, may promote stem cell growth and prevent differentiation.

[0187] The stem cells may be genetically modified at any stage withmarkers so that the markers are carried through to any stage ofcultivation. The markers may be used to purify the differentiated orundifferentiated stem cell population at any stage of cultivation.

[0188] Genetic construct may be inserted to undifferentiated ordifferentiated cells at any stage of cultivation. The geneticallymodified cells may be used after transplantation to carry and expressgenes in target organs in the course of gene therapy.

[0189] Progress of the stem cells and their maintenance in adifferentiated or undifferentiated stage may be monitored in aquantitative fashion by the measurement of stem cell specific secretedproducts into the culture medium or in fixed preparations of the cellsusing ELISA or related techniques. Such stem cell specific productsmight include the soluble form of the CD30 antigen or the GCTM-2 antigenor they may be monitored as described above using cell markers or geneexpression.

[0190] In another aspect of the invention there is provided a method ofinducing somatic differentiation of stem cells in vitro into progenitorcells said method comprising:

[0191] obtaining undifferentiated stem cells; and

[0192] providing a differentiating signal under conditions which arenon-permissive for stem cell renewal, do not kill cells and/or inducesunidirectional differentiation toward extraembryonic lineages.

[0193] The undifferentiated cell lines of the present invention may becultured indefinitely until a differentiating signal is given.Preferably, they are cultured under the conditions described above.

[0194] In the presence of a differentiation signal, undifferentiated EScells in the right conditions will differentiate into derivatives of theembryonic germ layers (endoderm, mesoderm and ectoderm) such as neurontissue, and/or extraembryonic tissues. This differentiation process canbe controlled.

[0195] This method is useful for directing stem cells to differentiatetoward a somatic lineage. Furthermore, the method allows theestablishment of a pure preparation of progenitor cells from a desiredlineage and the elimination of unwanted differentiated cells from otherlineages. The method facilitates the establishment of a pure somaticprogenitor cell line.

[0196] The method may be used to derive an enriched preparation of avariety of somatic progenitors such as but not limited to mesodermalprogenitors (such as hemangioblast or hematopoietic stem cells) andneural progenitors. Preferably the method is used to derive neuralprogenitors.

[0197] Conditions for obtaining differentiated cultures of somatic cellsfrom embryonic stem cells are those which are non-permissive for stemcell renewal, but do not kill stem cells or drive them to differentiateexclusively into extraembryonic lineages. A gradual withdrawal fromoptimal conditions for stem cell growth favours somatic differentiation.The stem cells are initially in an undifferentiated state and can beinduced to differentiate.

[0198] In a preferred embodiment of the present invention, there isprovided a method of inducing somatic differentiation of stem cells invitro into progenitor cells, said method comprising:

[0199] obtaining undifferentiated stem cells; and

[0200] culturing said cells for prolonged periods and at high density ona fibroblast feeder cell layer to induce differentiation.

[0201] In another preferred embodiment of the present invention, thereis provided a method of inducing somatic differentiation of stem cellsin vitro into progenitor cells, said method comprising:

[0202] obtaining undifferentiated stem cells; and

[0203] transferring said cells into serum free media to inducedifferentiation.

[0204] The stem cells may be undifferentiated stem cells and derivedfrom any source or process which provides viable undifferentiated stemcells. The methods described above for retrieving stem cells fromembryos is most preferred.

[0205] In these preferred aspects, the conditions of culturing the cellsat high density on a fibroblast feeder cell layer or transferring to aserum free medium are intended to be non-permissive for stem cellrenewal or cause uni-directional differentiation toward extraembryoniclineages.

[0206] Generally the presence of a fibroblast feeder layer will maintainthese cells in an undifferentiated state. This has been found to be thecase with the cultivation of mouse and human ES cells. However, withoutbeing restricted by theory, it has now become evident that the type andhandling of the fibroblast feeder layer is important for maintaining thecells in an undifferentiated state or inducing differentiation of thestem cells.

[0207] Suitable fibroblast feeder layers are discussed above.

[0208] Somatic differentiation in vitro of the ES cell lines is afunction of the period of cultivation following subculture, the densityof the culture, and the fibroblast feeder cell layer. It has been foundthat somatic differentiation may be detected as early as the first weekafter subculture and is morphologically apparent and demonstrable byimmunochemistry approximately 14 days following routine subcultivationas described above in areas of the colony which are remote from directcontact with the feeder cell layer (in contrast to areas adjacent to thefeeder cell layer where rapid stem cell growth is occurring such as theperiphery of a colony at earlier time points after subcultivation), orin cultures which have reached confluence. Depending upon the method ofpreparation and handling of the mouse embryo fibroblasts, the mousestrain from which the fibroblasts are derived, and the quality of aparticular batch, stem cell renewal, extraembryonic differentiation orsomatic differentiation may be favoured.

[0209] Once a suitable fibroblast cell line is selected, it may be usedas a differentiation inducing fibroblast feeder layer to induce theundifferentiated stem cells to differentiate into a somatic lineage ormultiple somatic lineages. These may be identified using markers or geneexpression as described above. Preferably the fibroblast feeder layerdoes not induce extraembryonic differentiation and cell death.

[0210] The modulation of stem cell growth by appropriate use offibroblast feeder layer and manipulation of the culture conditions thusprovides an example whereby somatic differentiation may be induced invitro concomitant with the limitation of stem cell renewal without theinduction of widespread cell death or extraembryonic differentiation.

[0211] Other manipulations of the culture conditions such as culturingin various compositions of serum free medium may be used to arrest stemcell renewal without causing stem cell death or unidirectionalextraembryonic differentiation, thereby favouring differentiation ofsomatic cells.

[0212] Differentiation may also be induced by culturing to a highdensity in monolayer or on semi-permeable membranes so as to createstructures mimicing the postimplantation phase of human development, orany modification of this approach. Cultivation in the presence of celltypes representative of those known to modulate growth anddifferentiation in the vertebrate embryo (eg. endoderm cells or cellsderived from normal embyronic or neoplastic tissue) or in adult tissues(eg. bone marrow stromal preparation) may also induce differentiation,modulate differentiation or induce maturation of cells within specificcell lineage so as to favour the establishment of particular celllineages.

[0213] Chemical differentiation may also be used to inducedifferentiation. Propagation in the presence of soluble or membranebound factors known to modulate differentiation of vertebrate embryoniccells, such as bone morphogenetic protein-2 or antagonists of suchfactors, may be used.

[0214] Applicants have found that Oct-4 is expressed in stem cells anddown-regulated during differentiation and this strongly indicates thatstem cell selection using drug resistance genes driven by the Oct-4promoter will be a useful avenue for manipulating human ES cells.Directed differentiation using growth factors, or the complementarystrategy of lineage selection coupled with growth factor enhancementcould enable the selection of populations of pure committed progenitorcells from spontaneously differentiating cells generated as describedhere.

[0215] Genetic modification of the stem cells or further modification ofthose genetically modified stem cells described above may be employed tocontrol the induction of differentiation. Genetic modification of thestem cells so as to introduce a construct containing a selectable markerunder the control of a promoter expressed only in specific celllineages, followed by treatment of the cells as described above and thesubsequent selection for cells in which that promoter is active may beused.

[0216] Once the cells have been induced to differentiate, the variouscell types, identified by means described above, may be separated andselectively cultivated. Preferably neural progenitor cells are selected.These progenitors are capable of differentiating into neuron cellsand/or glial cells. More preferably, they will differentiate into neuroncells and/or glial cells in the absence of other differentiated cellssuch as those from the extra embryonic lineage.

[0217] Selective cultivation means isolation of specific lineages ofprogenitors or mature differentiated cells from mixed populationspreferably appearing under conditions unfavourable for stem cell growthand subsequent propagation of these specific lineages. Selectivecultivation may be used to isolate populations of mature cells orpopulations of lineage specific committed progenitor cells. Isolationmay be achieved by various techniques in cell biology including thefollowing alone or in combination: microdissection; immunologicalselection by labelling with antibodies against epitopes expressed byspecific lineages of differentiated cells followed by direct isolationunder fluorescence microscopy, panning, immunomagnetic selection, orselection by flow cytometry; selective conditions favouring the growthor adhesion of specific cell lineages such as exposure to particulargrowth or extracellular matrix factors or selective cell-cell adhesion;separation on the basis of biophysical properties of the cells such asdensity; disaggregation of mixed populations of cells followed byisolation and cultivation of small clumps of cells or single cells inseparate culture vessels and selection on the basis of morphology,secretion of marker proteins, antigen expression, growth properties, orgene expression; lineage selection using lineage specific promoterconstructs driving selectable markers or other reporters.

[0218] The derivation of neural progenitors from ES cells, and evenfurther more, the establishment of a pure neural progenitor cell line isdescribed below as proof of the above principles. The followingdescription is illustrative of neural progenitor cells as somatic cellsdifferentiated from stem cells and should not be taken as a restrictionon the generality of the invention. It should be noted that the methodmay be used to derive an enriched preparation of a variety of somaticprogenitors such as but not limited to mesodermal progenitors such ashemangioblast or hematopoietic stem cells or neural progenitors.Transdifferentiation to non-neuronal phenotypes from neural progenitorsis within the scope of the present invention and may result inhematopoietic, endothelial, embryonic endoderm and ectodermal cells.

[0219] The establishment of neural progenitor cells from embryonic stemcells and more preferably a pure preparation of neural progenitor cellsand even more preferably a neural progenitor cell line may be achievedby any one or combination of the following approaches.

[0220] In one preferred approach, somatic differentiation of ES cells isinduced by prolonged culture of ES cells to high density on anappropriate fibroblast feeder layer that prevents unidirectionaldifferentiation towards extraembryonic lineage and promotes somaticdifferentiation. Once the cells have been induced to differentiatetoward somatic lineages, areas which are destined to give rise toclusters of mainly neural progenitor cells may be identified based oncharacteristic morphological features as described above. The size anddemarcation of these areas may be enhanced by replacing the growthmedium with serum free medium supplemented with EGF and bFGF. The areasare separated mechanically and replated in serum-free medium, whereuponthey form spherical structures.

[0221] Any serum free medium may be used. Preferably NS-A (Euroclone) orDMEM/F12 (Gibco) is used. More preferably NS-A or DMEM/F12 supplementedwith N2 or B27 (Gibco) is used. Most preferably DMEM/F12 supplementedwith B27 is used.

[0222] In the presence of an appropriate supplement of growth factorssuch as but not limited to, EGF and basic FGF to the serum free medium,the neural progenitors may be cultivated and expanded to establish acell line. The growth factors inhibit further differentiation of theprogenitor cells and promote their proliferation.

[0223] The culture in the serum free medium and preferably growthfactors is selective and therefore limits prolonged proliferation ofother types of differentiated cells such as the progeny of theextraembryonic lineage that may coexist in the culture. Therefore thecultivation in these selective conditions may be used to establish anenriched cell line of neural progenitors.

[0224] The progenitors may be cultured as spheres or as a monolayer.Subculturing may be conducted mechanically. Scraping is preferred topropagate monolayer cultures. However, any mechanical method such astituration or cutting may be used to subculture the spheres. Mostpreferably the spheres are sliced into smaller clumps. The progenitorsmay be expanded to produce a large number of cells.

[0225] In another preferred approach, the method involves the transferof undifferentiated stem cells into culture conditions that on one handdirect differentiation toward a desired somatic lineage, which is theneural lineage in this case, while on the other hand are selective andtherefore limit both the differentiation toward unwanted lineages (suchas extraembryonic lineages) as well as the survival of differentiatedcells from these lineages. Such culture conditions include the transferinto serum free media (as described above) that may be supplemented withgrowth factors including but not limited to bFGF and EGF. The serum freemedia promotes differentiation towards the neuroectodermal lineage (andpossibly other non-neural lineages such as mesoderm). The serum freemedia may limit the growth and survival of unwanted cells such as thosefrom the extraembryonic lineages.

[0226] In a further preferred embodiment of the invention, the methodallows the establishment of a pure progenitor cell line from the desiredlineage.

[0227] Growth factors that are added to the medium may promote theproliferation and the cultivation of the desired somatic progenitorssuch as neural progenitors. The selective culture conditions furthereliminate during cultivation, unwanted differentiated cells from otherlineages such as extraembryonic lineages. The method may be used toderive a pure preparation and/or a pure cell line of a variety ofsomatic progenitors including, but not limited to, neural progenitor,mesodermal progenitors such as hemangioblast or hematopoietic stem cellsand progenitors of the endodermal lineage.

[0228] Preferably, in the derivation of an enriched cell line of neuralprogenitors, clumps of undifferentiated stem cells may be transferredinto plastic tissue culture dishes containing serum free medium. Theserum free medium induces the differentiation of the ES cells initiallytowards ectoderm and then towards the neuroectodermal lineage.

[0229] Any serum free medium may be used. Preferably NS-A medium(Euroclone) or DMEM/F12 is used. More preferably the serum free mediumis supplemented with N2 or B27 (Gibco). Most preferably the medium isDMEM/F12 supplemented with B27. The clusters of undifferentiated stemcells turn into round spheres within approximately 24 hours aftertransfer (FIG. 9).

[0230] The serum free medium may be further supplemented with basic FGFand EGF to promote proliferation of neural progenitors in anundifferentiated state. The progenitors may be cultivated under theseconditions for prolonged periods. The selective conditions that areinduced by the serum free medium and growth factors result in a gradualpurification and elimination of other differentiated cell types duringcultivation.

[0231] The progenitors may be cultured as spheres or as a monolayer.Subculturing may be conducted mechanically. Scraping is preferred topropagate monolayer cultures. Any mechanical method known to the skilledaddressee such as tituration or cutting may be used to subculture thespheres. Most preferably the spheres are sliced into smaller clumps. Theprogenitors may be expanded to produce a large number of cells.

[0232] The progenitors that are generated directly from undifferentiatedstem cells have similar properties to the neural progenitors that aregenerated from differentiating stem cells colonies. They express thesame markers of primitive neuroectoderm and neural progenitor cells,such as N-CAM, polysialyated N-CAM, the intermediate filament proteinnestin, Vimentin and the transcription factor Pax-6. They do not expressthe transcriptional factor oct-4. They have a similar growth potential.They generate differentiated neural cells with similar morphology andmarker expression after plating on appropriate substrate and withdrawalof growth factors.

[0233] In another aspect of the invention, there is provided a method ofinducing somatic cells from embryonic stem cell derived somaticprogenitors, said method comprising:

[0234] obtaining a source of embryonic stem cell derived somaticprogenitor cells;

[0235] culturing the progenitor cells on an adhesive substrate; and

[0236] inducing the cells to differentiate to somatic cells underconditions which favour somatic differentiation.

[0237] The source of embryonic stem cell derived progenitor cells may befrom any source. However, they are preferably established by the methodsdescribed above. Preferably, the cells are grown in the presence of aserum-free media and growth factor.

[0238] The somatic cells may preferably be neurons, or glial cellsincluding astrocytes or oligodendrocyte cells. Preferably, the somaticprogenitors are neural progenitors.

[0239] Any adhesive substrate may be used. More preferably,poly-D-lysine and laminin or poly-D-lysine and fibronectin are used.

[0240] Induction of somatic cells is preferably achieved by withdrawinggrowth factors from the media. However, other acceptable methods ofinduction may be used. These may include:

[0241] culturing the undifferentiated cells for prolonged periods and athigh density to induce differentiation;

[0242] culturing the cells in serum free media;

[0243] culturing the cells on a differentiation inducing fibroblastfeeder layer and wherein said fibroblast feeder layer does not induceextra embryonic differentiation and cell death;

[0244] culturing to a high density in monolayer or on semi-permeablemembrane so as to create structures mimicing the postimplantation phaseof human development; or

[0245] culturing in the presence of a chemical differentiation factorselected from the group including bone morphogenic protein-2 orantagonists thereof.

[0246] For inducing neurons, it is preferred to further usepoly-D-lysine and laminin.

[0247] Upon plating of neural progenitors on an appropriate substratesuch as poly-D-lysine and laminin, and withdrawal of growth factors fromthe serum free medium, differentiated cells grow out of the spheres as amonolayer and acquire morphology of mature neurons and expression ofmarkers such as the 160 kDa neurofilament protein, Map-2AB,synaptophysin, Glutamate, GABA, serotonin, tyrosine hydroxylase, GABAbiosynthesis and receptor subunits characteristic of GABA minergicneurons (GABA Aα2) which are characteristic of mature neurons.

[0248] In a preferred embodiment, the method for inducing neuronsfurther includes culturing the somatic progenitor cells, preferablyundifferentiated neural progenitor cells, or differentiating neuronalprogenitors in the presence of retinoic acid.

[0249] Retinoic acid has been found to further induce differentiationtoward mature neurons.

[0250] Accordingly, there is provided a mature neuron cell prepared bythe method described herein. The mature neuron cell may be characterizedin that it expresses 160 kDa neuro-filament protein, MAP2ab, glutamate,synaptophysin, glutamic acid decarboxylase (GAD), tyrosine hydroxylase,GABA and serotonin.

[0251] The establishment of oligodendrocyte and astrocyte cellsindicates the potential of the neural precursors to differentiatetowards the glial lineage.

[0252] For inducing of glial cells including astrocytes andoligodendrocyte progenitors, it is preferred to use poly-D-lysine andfibronectin. Fibronectin is significantly more potent than laminin forthe induction of differentiation towards the glial lineage.

[0253] In a preferred embodiment, the method for inducing glial cellsfurther includes culturing the somatic progenitor cells, preferablyundifferentiated neural progenitor cells, in the presence of PDGF-AA andbasic FGF.

[0254] In yet another preferred embodiment, the method for inducingglial cells further includes culturing the somatic progenitor cells,preferably undifferentiated neural progenitor cells, in the presence ofT3. The cells may be then grown in the absence of growth factor.

[0255] The glial cells may be selected from astrocytes oroligodendrocytes. The oligodendrocytes derived from the methods of thepresent invention may be identified and characterized by RNA transcriptsof MBP, plp and dm-20 or by immunostaining for O4 and NG2.

[0256] Culture in serum free medium supplemented with b-FGF and PDGF-AAmay direct the neural progenitors to turn into glial progenitors andinduce the expansion of glial progenitors. This is followed by platingthe progenitors on poly-D-lysine and fibronectin and further culture inthe presence of the growth factors and T3 followed by culture in thepresence of T3 without growth factor supplementation. Without beinglimited by theory, it is postulated that the growth factors such as bFGFand PDGF-AA facilitate proliferation and spreading of the glialprogenitors, fibronectin further induces differentiation towards theglial lineage and T3 induce the differentiation toward and along theoligodendrocyte lineage.

[0257] In another aspect, differentiation into glial cells includingastrocyte and oligodendrocyte cells is induced by plating the neuralprogenitors on poly-D-lysine and fibronectin and culturing them in theserum free medium supplemented with EGF, b-FGF and PDGF-AA. The growthfactors may then be removed and the cells further cultured in thepresence of T3.

[0258] In yet another aspect, the invention provides differentiatedsomatic cells including neural, neural progenitor cells, neuronal and/orglial cells prepared by the methods of the present invention. The glialcells include astrocytes or oligodendrocytes. The oligodendrocytesderived by the methods of the present invention may be characterized byRNA transcripts of MBP, plp and dm-20 or by immunostaining for O4 andNG2.

[0259] The progenitor cells that are derived by the method that isdescribed above may be used to generate differentiated cells from otherlineages. The spheres of progenitors may include in addition to neuralprogenitors more primitive cells such as primitive ectodermal cells orprogenitor cells of other lineages such as the hemangioblast endothelialor hematopoietic stem cells, embryonic endodermal cells and ectoderm. Bymanipulation of the culture conditions these primitive cells maygenerate all somatic cell types.

[0260] Expression of mesodermal markers such as flk-1, AC-133 and CD-34,embryonic endodermal markers such as transferin, amylase and α1 antitrypsin and the epidermal marker keratin has been demonstrated in thehuman ES derived progenitor cell preparation. This may indicate thepresence of primitive cells from non-neural lineages such as thehemangioblast cell or hematopoietic stem cell within the neuralprogenitors preparation. Alternatively it may be that the primitiveneural progenitors within the spheres express these markers. Theexpression of the markers may indicate the possible high plasticity ofthe neural progenitors to transdifferentiate into cells of otherlineages.

[0261] The present invention provides a method that generates an invitro and in vivo model of controlled differentiation of ES cellstowards the neural lineage. The model, and the cells that are generatedalong the pathway of neural differentiation may be used for the study ofthe cellular and molecular biology of human neural development, for thediscovery of genes, growth factors, and differentiation factors thatplay a role in neural differentiation and regeneration. The model, andthe cells that are generated along the pathway of neural differentiationmay be used for drug discovery and for the development of screeningassays for teratogenic, toxic and neuroprotective effects.

[0262] In a further aspect of the invention, there is provided a methodof producing large quantities of differentiated and undifferentiatedcells. It is intended to mean that these cells can be propagated,expanded and grown in cell culture.

[0263] In yet another aspect, the present invention provides a method ofproducing an enriched preparation of human ES derived neural progenitorcells, said method comprising:

[0264] obtaining an undifferentiated human embryonic stem cell asdescribed herein;

[0265] inducing somatic differentiation of the embryonic stem cell to aneural progenitor cell by a method described herein;

[0266] identifying a neural progenitor cell by expressed markers ofprimitive neuroectoderm and neural stem cells such as N-CAM,polysialyated N-CAM, A2B5, intermediate filament proteins such as nestinand vimentin and the transcription factor Pax-6; and

[0267] culturing the neural progenitor cells to promote proliferationand propagation.

[0268] The neural progenitor cells will grow as spheres or monolayerspreferably in serum free media. A suitable media is DMEM/F12supplemented with growth factors selected from the group including B27,EGF and bFGF.

[0269] Further enrichment of the preparation may be achieved by furthercultivation in new media that includes transferring the clumps of cellsinto new media.

[0270] In a further aspect of the invention there is provided a methodto dis-aggregate the spheres into single cell suspensions.Dis-aggregation by using digestion with trypsin or dispase may beineffective. Dis-aggregation may be accomplished by digestion withpapain combined with mechanical tituration.

[0271] In another aspect of the invention, there is provided a method oftransplanting ES derived neural progenitor spheres, said methodcomprising:

[0272] disaggregating the spheres; and

[0273] injecting the disaggregated spheres into a living host.

[0274] Disaggregation of the spheres may be conducted in any way toseparate the cells either to small clumps or single cells. Ideally,trypsin or dispase are not used. Mechanical disaggregation or titurationmay be adopted to separate the cells prior to injection. Alternativelythe spheres may be disaggregated by digestion with papain preferablycombined with mechanical tituration.

[0275] Injection may be conducted in any manner so as to introduce thecells into the nervous system of the host. Preferably the cells areintroduced into a specific site in the nervous system. Any method may beused to introduce the cells into a specific location. Preferably, thecells are injected using a micro-glass pipette (300 micron outerdiameter) connected to a micro-injector (Narishige, Japan). The glasspipette may be covered by a plastic sleeve that will limit the depth ofpenetration into the host nervous system. The cells may be also injectedby a hamilton syringe into predetermined depth using a stereotaxicdevice. Any stereotaxic injection method may be suitable.

[0276] The volume that is injected and the concentration of cells in thetransplanted solution depend on the indication for transplantation, thelocation in the nervous system and the species of the host. Preferably 2microliters with 25,000-50,000 cells per microliter are injected to thelateral cerebral ventricles of newborn rats or mice.

[0277] In another aspect of the invention there is provided a neuralprogenitor cell capable of transplantation into a host nervous systemsaid cell characterised by establishing a stable graft and contributingin the histogenesis of a living host.

[0278] In another aspect of the present invention there is provided amethod of inducing somatic cells in vivo from embryonic stem cellderived somatic progenitors, said method comprising:

[0279] obtaining a source of embryonic stem cell derived somaticprogenitor cells, preferably prepared by the methods described herein;and

[0280] transplanting the somatic progenitors into a host to inducedifferentiation to somatic cells.

[0281] The transplanting may be conducted by any of the methodsdescribed herein.

[0282] When engrafted into a developing nervous system, the progenitorcells of the present invention will participate in the processes ofnormal development and will respond to the host's developmental cues.The engrafted progenitor cells will migrate along established migratorypathways and will spread widely into disseminated areas of the nervoussystem. The transplanted cells will respond to host environmentalsignals, differentiate in a temporally and regionally appropriate mannerinto progeny from both the neuronal and glial lineages in accord to theregion's stage of development and in concert with the host developmentalprogram. The engrafted neural progenitor cell is capable ofnon-disruptive intermingling with the host neural progenitors as well asdifferentiated cells. The transplanted cells can replace specificdeficient neuronal or glial cell populations, restore defectivefunctions and can express foreign genes in a wide distribution.

[0283] In a further aspect of the invention the ES derived neuralprogenitor cells or their differentiated progeny may be transplantedinto the developed nervous system. They can form a stable graft, migratewithin the host nervous system, intermingle and interact with the hostneural progenitors and differentiated cells. They can replace specificdeficient neuronal or glial cell populations, restore deficientfunctions and activate regenerative and healing processes in the host'snervous system. In an even further aspect of the invention thetransplanted cells can express foreign genes in the host's nervoussystem.

[0284] Preferably the stable graft is a graft established in the centralnervous system or the peripheral nervous system. The stable graft mayestablish in the brain. The neural progenitor cells of the presentinvention have been shown by the applicants to differentiate to matureneurons characterized by their ability to express 160 kDa neuro-filamentprotein, MAP2ab, glutamate, synaptophysin, glutamic acid decarboxylase(GAD), tyrosine hydroxylase, GABA and serotonin. The neural progenitorcells of the present invention have been also shown by the applicants todifferentiate to astrocyte and oligodendrocyte characterized by RNAtranscripts of GFAP, MBP, plp and dm-20 or by immunostaining for GFAP,O4 and NG2.

[0285] The ability to differentiate to the various somatic cell types isparticularly useful for modifying the nervous system for replacingdeficient neuronal or glial cell populations, restoring deficientfunctions of the system, or activating regenerative and healingprocesses in the nervous system.

[0286] In a further aspect of the invention the progenitor cells aregrafted into other organs such as but not limited to the hematopoieticsystem where they trans-differentiate and form a stable functionalgraft.

[0287] More preferably the spheres are ES derived human neuralprogenitor spheres which are transplanted into the living host.

[0288] In yet another aspect of the present invention, there is provideda method of treating a mental condition by replenishing a cellpopulation in the brain, said method comprising:

[0289] obtaining a source of embryonic stem cell derived somaticprogenitor cells, preferably prepared by the methods described herein;and

[0290] transplanting the somatic progenitors into a host to promotetheir differentiation to somatic cells.

[0291] Preferably, they are differentiated from neural lineages selectedfrom neurons, astrocytes and oligodendrocyte.

[0292] Preferably the mental condition is selected from the groupincluding alzheimers disease, and other mental conditions causingdementia. It is shown that mature neurons of the present inventionexpress glutamate, TH, GABA and serotonin. Serotoninergic neurons mayhave a role in the pathogenesis and treatment of mental conditions.

[0293] In a further aspect of the invention there is provided a neuralprogenitor cell, a neuronal cell and/or a glial cell that may be usedfor cell therapy in a variety of pathological conditions including butnot limited to neurodegenerative disorders, vascular conditions,autoimmune disorders, congenital disorders, trauma and others.

[0294] In a further aspect of the invention there is provided a neuralprogenitor cell, a neuronal cell and/or a glial cell that may be usedfor gene therapy. Genetically manipulated neural progenitor cells orneuronal cell or glial cells may be used after transplantation as avector to carry and express desired genes at target organs.

[0295] In another aspect of the present invention, there is provided acommitted progenitor cell line. The progenitor cell line may be expandedto produce large quantities of progenitor cells, neural progenitorcells, neuronal cells, mature neuronal cells and glial cells.

[0296] In another aspect of the invention, there are provided committedneural progenitor cells capable of self renewal or differentiation intoone or limited number of somatic cell lineages, as well as maturedifferentiated cell produced by the methods of the present invention.

[0297] Expansion of the committed progenitor cells may be useful whenthe number of progenitors that may be derived from ES cells is limited.In such a case, expansion of the progenitors may be useful for variousapplications such as the production of sufficient cells fortransplantation therapy, for the production of sufficient RNA for genediscovery studies etc. For example, by using the techniques describedabove, expansion of progenitor cells from ten spheres for ten passagesmay generate 50×10⁶ cells that would be sufficient for any application.

[0298] These observations on cells of the neural lineage establish theprinciple that by using the techniques described, committed progenitorcells may be isolated, from embryonic stem cell cultures propagated,expanded, enriched and further induced to produce fully differentiatedcells.

[0299] In a further aspect of the invention, there is provided a methodof producing large quantities of differentiated and undifferentiatedcells.

[0300] In another aspect there is provided a differentiated committedprogenitor cell line that may be cultivated for prolonged periods andgive rise to large quantities of progenitor cells and fullydifferentiated cells.

[0301] The neural progenitor cells or other committed progenitor cellsderived by the method described above may be used to generatedifferentiated cells from other lineages by transdifferentiation.

[0302] In another aspect there is provided a differentiated committedprogenitor cell line capable of differentiation into mature neuronsand/or glial cells. Preferably the progenitor cell is a neuralprogenitor cell.

[0303] In another aspect there is provided an undifferentiated cell linecapable of differentiation into neural progenitor cells produced by themethod of the present invention.

[0304] Specific cell lines HES-1 and HES-2 were isolated by theprocedures described above and have the properties described above.

[0305] In another aspect of the invention there is provided a cellcomposition including a human differentiated or undifferentiated cellcapable of differentiation into neural progenitor cells preferablyproduced by the method of the present invention, and a carrier.

[0306] The carrier may be any physiologically acceptable carrier thatmaintains the cells. It may be PBS or ES medium.

[0307] The differentiated or undifferentiated cells may be preserved ormaintained by any methods suitable for storage of biological material.Vitrification of the biological material is the preferred method overthe traditional slow-rate freezing methods.

[0308] Effective preservation of ES cells is highly important as itallows for continued storage of the cells for multiple future usage.Although traditional slow freezing methods, commonly utilised for thecryo-preservation of cell lines, may be used to cryo-preserveundifferentiated or differentiated cells, the efficiency of recovery ofviable human undifferentiated ES cells with such methods is extremelylow. ES cell lines differ from other cell lines since the pluripotentcells are derived from the blastocyst and retain their embryonicproperties in culture. Therefore, cryo-preservation using a method whichis efficient for embryos is most appropriate. Any method which isefficient for cryo-preservation of embryos may be used. Preferably,vitrification method is used. More preferably the Open Pulled Straw(OPS) vitrification method previously described by Vajta, G. et al(1998) Molecular Reproduction and Development, 51, 53-58, is used forcryopreserving the undifferentiated cells. More preferably, the methoddescribed by Vajta, G. et al (1998) Cryo-Letters, 19, 389-392 isemployed. Generally, this method has only been used for cryopreservingembryos.

[0309] The committed progenitor cell line is efficiently recovered fromcryopreservation using the traditional slow rate cooling method.

[0310] The differentiated or undifferentiated cells may be used as asource for isolation or identification of novel gene products includingbut not limited to growth factors, differentiation factors or factorscontrolling tissue regeneration, or they may be used for the generationof antibodies against novel epitopes. The cell lines may also be usedfor the development of means to diagnose, prevent or treat congenitaldiseases.

[0311] Much attention recently has been devoted to the potentialapplications of stem cells in biology and medicine. The properties ofpluripotentiality and immortality are unique to ES cells and enableinvestigators to approach many issues in human biology and medicine forthe first time. ES cells potentially can address the shortage of donortissue for use in transplantation procedures, particularly where noalternative culture system can support growth of the required committedstem cell. ES cells have many other far reaching applications in humanmedicine, in areas such as embryological research, functional genomics,identification of novel growth factors, and drug discovery, andtoxicology.

[0312] While the potential applications of neural stem cells derivedfrom adult or embryonic CNS are considerable, there may be realadvantages to neural progenitor cells derived from ES cell cultures.

[0313] ES cell lines derived from a patients' own tissue via somaticcell nuclear transfer would produce neuronal precursors which are aprecise match to the recipients own tissue and might therefore be moresuitable for grafting.

[0314] Moreover the use of nuclear transfer to yield ES cells fromindividuals with specific genetic predispostions to certain diseases ofthe CNS could provide a powerful tool for the generation of in vitromodels for disease pathogenesis.

[0315] It is quite likely that neural precursors generated from ES cellcultures may demonstrate a greater growth or developmental potentialthan committed progenitors from fetal or adult CNS.

[0316] There is a huge range of cell types within the adult CNS, andwhile it is clear that ES cells can give rise to any of these in themouse, it is not clear that neural stem cells can do so.

[0317] ES derived neural progenitors may allow the study of early stagesof the process of neurogenesis, and thereby provide important clues fordiscovery of novel factors enhancing tissue regeneration, or novel stemcell intermediates which might be more facile at replacing damagedtissue.

[0318] It may be that the frequency of homologous recombination in EScells is much higher than that in neural stem cells, and therefore thatthe only practical route for introducing targetted genetic modificationsinto human neural tissue-either for generation of disease models invitro or for types of gene therapy-lies in the reproducible generationand isolation of neural progenitors from genetically modified embryonicstem cells.

[0319] The present invention will now be more fully described withreference to the following examples. It should be understood, however,that the description following is illustrative only and should not betaken in any way as a restriction on the generality of the inventiondescribed above.

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[0331] 12. Fong C. Y., and Bongso A. Comparison of human blastulationrates and total cell number in sequential culture media with and withoutco-culture. Hum. Reprod. 14, 774-781 (1999).

[0332] 13. Fong C. Y. et al. Ongoing pregnancy after transfer ofzona-free blastocysts: implications for embryo transfer in the human.Hum. Reprod. 12, 557-560 (1997).

[0333] 14. Solter D., and Knowles, B. Immunosurgery of mouse blastocyst.Proc. Natl. Acad. Sci. U.S.A. 72, 5099-5102 (1975).

[0334] 15. Vajta G, Holm P, Kuwayama M, Both P J, Jacobsen H, Greve T,Callesen H. Open pulled straw (OPS) vitrification: A new way to reducecryoinjuries of bovine ova and embryos. Molecular Reproduction andDevelopment 1998, 51: 53-58.

[0335] 16. Vajta G, Lewis I M, Kuwayama M, Greve T, Callesen H. Sterileapplication of the opened pulled straw (OPS) vitrification method.Cryo-Letters 1998, 19: 389-392.

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[0337] 18. Li, M., Pevny, L., Lovell-Badge, R., and Smith, A. Generationof purified neural precursors from embryonic stem cells by lineageselection. Curr. Biol. 8, 971-974 (1998).

[0338] 19. Svensden, C N and Smith A G New prospects for human stem-celltherapy in the nervous system. Trends in Neurosci 22: 357, 1999.

[0339] 20. Kleinsmith U and Pierce G B. Multipotentiality of singleembryonal carcinoma cells. Cancer Res. 1964; 24: 797-842.

[0340] 21. Stevens (1983)

[0341] 22. Martin G R. Teratocarcinomas and mammalian embryogenesis.Science 1980; 209: 768.

[0342] 23. Matsui Y, Zsebo K, Hogan B L M. Derivation of pluripotentembryonic stem cells from murine primordial germ cells in culture. Cell1992; 70: 841-847.

[0343] 24. Andrews P W. Human teratocarcinoma. Biochim. Biophys. Acta1988; 948:17-36.

[0344] 25. Thompson S, Stern P L, Webb M, et al.,. Cloned human teratomacells differentiate into neuron-like cells and other cell types inretinoic acid. J Cell Sci. 1984; 72: 37-64.

[0345] 26. van Eijk M J T, van Rooijen M A, Modina S, Scesi L, FolkersG, van Tol H T A, Bevers M M, Fisher S R, Lewin H A, Shehu D, Galli C,de Vaureix C, Trounson A O, Mummery C L, and Gandolfi F. Molecularcloning, genetic mapping and developmental expression of bovine POU5F1.Biology of Reproduction 1999; 60: 1093-1103.

[0346] 27. Kukekov V G, Laywell E D, Suslov O et al. Multipotentstem/progenitor cells with similar properties arise from two neurogenicregions of adult human brain. Experimental Neurology 1999, 156: 333-344

[0347] 28. Uchida N, Buck D W, He D, Reitsma M J, Masek M, Phan T V,Tsukamoto A S, Gage F H, Weissman I L. Direct isolation of human centralnervous system stem cells. Proc Natl Acad Sci USA 2000, 97:14720-5.

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Experimental Protocols

[0355] 1. Derivation and Propagation of ES Cells

[0356] Fertilised oocytes were cultured to the blastocyst stage (day 6after insemination), in sequential media, according to a standardco-culture free protocol (Fong C. Y., and Bongso A. Comparison of humanblastulation rates and total cell number in sequential culture mediawith and without co-culture. Hum. Reprod. 14,774-781(1999)). After zonapellucida digestion by pronase (Sigma, St. Louis, Mo.) (Fong C. Y. etal. Ongoing pregnancy after transfer of zona-free blastocysts:implications for embryo transfer in the human. Hum. Reprod. 12, 557-560(1997)), ICM were isolated by immunosurgery (Solter D., and Knowles, B.Immunosurgery of mouse blastocyst. Proc. Natl. Acad. Sci. U.S.A. 72,5099-5102 (1975)) using anti-human serum antibody (Sigma) followed byexposure to guinea pig complement (Life Technologies, Gaithersburg,Md.). ICM were then cultured on mitomycin C mitotically inactivatedmouse embryonic fibroblast feeder layer (75,000 cells/cm2) in gelatinecoated tissue culture dishes. The culture medium consisted of DMEM(Gibco, without sodium pyruvate, glucose 4500 mg/L) supplemented with20% fetal bovine serum (Hyclone, Logan, Utah), 0.1 mMbeta-mercaptoethanol, 1% non essential amino acids, 2 mM glutamine, 50u/ml penicillin and 50(g/ml streptomycin (Life Technologies). During theisolation and early stages of ES cell cultivation, the medium wassupplemented with human recombinant leukemia inhibitory factor hLIF at2000 u/ml (Amrad, Melbourne, Australia). 6-8 days after initial plating,ICM like clumps were removed mechanically by a micropipette fromdifferentiated cell outgrowths and replated on fresh feeder layer. Theresulting colonies were further propagated in clumps of about 100 stemcell like cells, on mouse feeder layer, about every 7 days. The clumpswere either dissociated mechanically, or with a combined approach ofmechanical slicing followed by exposure to dispase (10 mg/ml, LifeTechnologies).

[0357] (a) Embryo Culture

[0358] Following insemination, embryos were cultured in droplets underpre-equilibrated sterile mineral oil in IVF-50 medium (Scandinavian 2medium) for 2 days.

[0359] A mixture 1:1 of IVF-50 and Scandinavian 2 medium (Scandinavian 2medium) was used in the third day.

[0360] From the forth day of culture, only Scandinavian 2 medium wasused to grow the cleavage stage embryos to blastocysts.

[0361] (b) Zona Pellucida Digestion

[0362] Zona pellucida digestion was performed at the expanded blastocyststage on day 6.

[0363] The digestion solution included Pronase (Sigma, TC tested) 10 uin PBS and Scandinavian 2 medium (1:1).

[0364] The embryos were incubated in pronase solution for 1-1.5 min,washed in Scandinavian 2 medium and incubated for 30 minutes. If thezona was not completely dissolved, the embryos were further incubated inpronase solution for 15 seconds.

[0365] (c) Human Stem Cell Culture

[0366] Human stem cells were grown on MMC treated fibroblasts' feederlayer. Fibroblasts were plated on gelatine treated dishes. A combinationof human and mouse derived fibroblasts were used at a density ofapproximately 25,000 and 70,000 cells per cm² respectively. Thefibroblasts were plated up to 48 hours before culture of the stem cells.Mouse fibroblasts only could also support the growth of the stem cells.However, while human fibroblasts could also support stem cells, theycreated an uneven and unstable feeder layer. Therefore, the humanfibroblasts were combined with the mouse fibroblasts to augment andachieve better support of growth and prevention of differentiation.

[0367] The medium that was used for the growth of human stem was DMEM(GIBCO, without sodium pyruvate, with glucose 4500 mg/L) supplementedwith 20% FBS (Hyclone, Utah) (-mercaptoethanol-0.1 mM (GIBCO), NonEssential Amino Acids-NEAA 1% (GIBCO), glutamine 2 mM.(GIBCO),penicillin 50u/ml, and streptomycin 50(g/ml (GIBCO). At the initialisolation of the stem cells the medium was supplemented by hLIF 2000u/ml. It was later shown that LIF was not necessary.

[0368] (d) Human Stem Cell Propagation

[0369] Following plating, the isolated ICM attached and was cultured for6 days. At that stage, a colony which included a clump of stem cells ontop of differentiated cells developed. The ICM clump was isolated andremoved mechanically by a micro-pipette with the aid of using Ca/Mg freePBS medium to reduce cell to cell attachments.

[0370] The isolated clump was replated on fresh human/mouse fibroblastfeeder layer. Following 2 weeks of culture, a colony with typicalmorphology of primate pluripotent stem cells developed. The stem cellswere further propagated in one of two methods. In both methods cellswhich appeared nondifferentiated were propagated in clumps of about 100cells every 5-7 days.

[0371] In the first method, Ca²+/Mg²+ free PBS medium was used to reducecell to cell attachments. Following about 15-20 minutes, cells graduallystart to dissociate and the desired size clumps can be isolated. Whencell dissociation is partial, mechanical dissociation using the sharpedge of the pipette assisted with cutting and the isolation of theclumps.

[0372] An alternative approach was performed by the combined use ofmechanical cutting of the colonies followed by isolation of thesubcolonies by dispase. Cutting of the colonies was performed in PBScontaining Ca and Mg. The sharp edge of micropipette was used to cut thecolonies to clumps of about 100 cells. The pipette was also used toscrape and remove differentiated areas of the colonies. The PBS was thenchanged to regular prequilibrated human stem cells medium containingdispase (Gibco) 10 mg/ml and incubated for 5-10 minutes (at 37(C., 5%CO2). As soon as the clumps were detached they were picked up by widebore micro-pipette, washed in PBS containing Ca and Mg and transferredto a fresh feeder layer.

[0373] e) Human Stem Cell Cryopreservation

[0374] Early passage cells were cryo-preserved in clumps of about 100cells by using the open pulled straw (OPS) vitrification method (Vajtaet al 1998) with some modifications. French mini-straws (250(I, IMV,L'Aigle, France) were heat-softened over a hot plate, and pulledmanually until the inner diameter was reduced to about half of theoriginal diameter. The straws were allowed to cool to room temperatureand were than cut at the narrowest point with a razor blade. The strawswere sterilised by gamma irradiation (15-25 K Gy). Two vitrificationsolutions (VS) were used. Both were based on a holding medium (HM) whichincluded DMEM containing HEPES buffer (Gibco, without sodium pyruvate,glucose 4500 mg/L) supplemented with 20% fetal bovine serum (Hyclone,Logan, Utah). The first VS (VS1) included 10% dimethyl sulfoxide (DMSO,Sigma) and 10% ethylene glycol (EG, Sigma). The second vitrificationsolution (VS2) included 20% DMSO, 20% EG and 0.5M sucrose. Allprocedures were performed on a heating stage at 37(C. 4-6 clumps of EScells were first incubated in VS1 for 1 minute followed by incubation inVS2 for 25 seconds. They were then washed in a 20(I droplet of VS2 andplaced within a droplet of 1-2(I of VS2. The clumps were loaded into thenarrow end of the straw from the droplet by capillary action. The narrowend was immediately submerged into liquid nitrogen. Straws were storedin liquid nitrogen. Thawing was also performed on a heating stage at 37°C. as previously described with slight modifications (Vajta et al 1998).Three seconds after removal from liquid nitrogen, the narrow end of thestraw was submerged into HM supplemented with 0.2M sucrose. After 1minute incubation the clumps were further incubated 5 minutes in HM with0.1 M sucrose and an additional 5 minutes in HM.

[0375] 2. Stem Cell Characterisation

[0376] Colonies were fixed in the culture dishes by 100% ethanol forimmuno-fluorescence demonstration of the stem cell surface markersGCTM-2, TRA 1-60 and SSEA-1, while 90% acetone fixation was used forSSEA-4. The sources of the monoclonal antibodies used for the detectionof the markers were as follows: GCTM-2, this laboratory; TRA 1-60, agift of Peter Andrews, University of Sheffield; SSEA-1 (MC-480) andSSEA-4 (MC-813-70), Developmental Studies Hybridoma Bank, Iowa, Iowa.Antibody localisation was performed by using rabbit anti-mouseimmunoglobulins conjugated to fluorescein isothiocyanate (Dako,Carpinteria, Calif.).

[0377] Alkaline phosphatase activity was demonstrated as previouslydescribed (Buehr M. and Mclaren A. Isolation and culture of primordialgerm cells. Methods Enzymol. 225, 58-76, (1993)). Standard G-bandingtechniques were used for karyotyping.

[0378] 3. Oct-4 Expression Studies

[0379] To monitor expression of Oct-4, RT-PCR was carried out oncolonies consisting predominantly of stem cells, or colonies which hadundergone spontaneous differentiation as described below. mRNA wasisolated on magnetic beads (Dynal AS, Oslo) following cell lysisaccording to the manufacturer's instructions, and solid-phase firststrand cDNA synthesis was performed using Superscript II reversetranscriptase (Life Technologies). The PCR reaction was carried outaccording to van Eijk et al. (1999), using the solid phase cDNA astemplate and Taq polymerase (Pharmacia Biotech, Hong Kong). OCT-4transcripts were assayed using the following primers:5′-CGTTCTCTTTGGAAAGGTGTTC (forward) and 3′-ACACTCGGACCACGTCTTTC(reverse). As a control for mRNA quality, beta-actin transcripts wereassayed using the same RT-PCR and the following primers:5′-CGCACCACTGGCATTGTCAT-3′ (forward), 5′-TTCTCCTTGATGTCACGCAC-3′(reverse). Products were analysed on a 1.5% agarose gel and visualisedby ethidium bromide staining.

[0380] 4. In-vitro Differentiation

[0381] Colonies were cultured on mitotically inactivated mouse embryonicfibroblasts to confluency (about 3 weeks) and further on up to 7 weeksafter passage. The medium was replaced every day. Alphafetoprotein andbeta human chorionic gonadotropin levels were measured in mediumconditioned by HES-1 and HES-2 at passage level 17 and 6 respectively.After 4-5 weeks of culture, conditioned medium was harvested 36 hoursafter last medium change, and the protein levels were determined by aspecific immunoenzymometric assays (Eurogenetics, Tessenderllo, Belgium)and a fluorometric enzyme immunoassay (Dade, Miami, Fla.) respectively.These compounds were not detected in control medium conditioned only byfeeder layer.

[0382] Differentiated cultures were fixed 6-7 weeks after passage(26-HES-1 and 9-HES-2) for immunofluorescence detection of lineagespecific markers. After fixation with 100% ethanol, specific monoclonalantibodies were used to detect the 68 kDa neurofilament protein(Amersham, Amersham U.K), and neural cell adhesion molecule (Dako).Muscle specific actin and desmin were also detected by monoclonalantibodies (Dako) after fixation with methanol/acetone (1:1). Antibodylocalisation was performed as described above.

[0383] 5. Teratoma Formation in Severe Combined Immunodeficient (SCID)mice

[0384] At the time of routine passage, clumps of about 200 cells with anundifferentiated morphology were harvested as described above, andinjected into the testis of 4-8 week old SCID mice (CB17 strain from theWalter and Eliza Hall Institute, Melbourne, Australia, 10-15clumps/testis). 6-7 weeks later, the resulting tumours were fixed inneutral buffered formalin 10%, embedded in paraffin and examinedhistologically after hematoxylin and eosin staining.

[0385] 6. Derivation and Culture of Neural Progenitors

[0386] Two approaches were developed for the derivation of neuralprecursors from human ES cells:

[0387] (a) Derivation of Neural Precursors from Differentiating ES Cells

[0388] Colonies of undifferentiated ES cells were continuously culturedon mouse embryonic fibroblasts for 2-3 weeks. The medium was changedevery day. Starting from the second week of culture and more commonly atthe third week, areas of tight small differentiated ES cells could beidentified in the colonies both by phase contrast microscopy as well asstereo microscopy. These areas tended to become well demarcated in thethird week of culture and had a typical uniform white gray opaqueappearance under dark field stereo-microscopy (FIG. 26). The size anddemarcation of these areas could be enhanced if after the first week ofculture, the serum containing medium was replaced with serum free mediumthat was supplemented with epidermal growth factor 20 ng/ml (EGF,Gibco), and basic fibroblast growth factor 20 ng/ml (bFGF, Gibco) andconsisted of DMEM/F12 (Gibco, Gaithersburg, Md.), B27 supplementation(1:50, Gibco), glutamine 2 mM (Gibco), penicillin 50 u/ml andstreptomycin 50 μg/ml (Gibco). Clumps of about 150 small tightly packedcells were dissected mechanically by a micropipette from these areas andwere transferred to plastic tissue culture dishes containing fresh serumfree medium (as detailed above), supplemented with EGF(20 ng/ml), andbasic FGF(20 ng/ml). The medium was supplemented with heparin 5 μg/ml(Sigma St. Louis, Mo.) in some of the experiments. The clusters of cellsturned into round spheres that were comprised of small tight cellswithin 24 hours after transfer. The spheres were sub-cultured aboutevery 7-21 days. The timing of subculture was determined according tothe size of the spheres. The diameter of spheres at the time ofsub-culture was usually above 0.5 mm. Each sphere was dissectedaccording to its size to 4 parts by two surgical blades (size 20,Swann-Morton, Sheffield, UK) to produce clumps with a maximal diameterbetween 0.3-0.5 mm. 50% of the medium was changed about every 3 days.

[0389] (b) Derivation of Neural Precursors from Undifferentiated ESCells

[0390] Colonies of undifferentiated ES cells were propagated on mouseembryonic fibroblasts as described above. Undifferentiated ES cells werepassaged in clumps of about 150-200 cells every 7 days. At the time ofroutine passage, clumps of about 200 ES cells were transferred toplastic tissue culture dishes containing the same serum free medium thatwas described in item 1 above. The clusters of cells turned into roundspheres within 24 hours after transfer. The spheres were sub-culturedabout every 7-21 days as described above. 50% of the medium was changedabout every 3 days.

[0391] (c) Characterization of the Growth and the Number of Cells in theSpheres

[0392] Growth of the progenitors was roughly evaluated by the increasein the number of spheres at each passage. The growth was also monitoredby serial measurements of the volume of 24 spheres. Individual sphereswere plated in twenty four well dishes (a sphere per well) and theirdiameter was evaluated every 7 days starting from the first passage (oneweek after derivation). The volume was calculated by using the volumeequation of a ball. The spheres were passaged every 7-21 days when thediameter of at least six spheres exceeded 0.5 mm. At each passage, sixspheres (diameter>0.5 mm) were sectioned into quarters that were platedindividually in a 24 well tissue culture dish. When the measurementsoccurred 7 days after passage, the sum of volumes of the daughterspheres was compared to the sum of volumes of the mother spheres.

[0393] The number of cells per sphere and its correlation with thediameter of the spheres was evaluated in a sample of spheres withvarious sizes. Each sphere was mechanically disaggregated into singlecells or by enzymatic (papain, Wortinington Biochemical Co, NJ)digestion that was followed by tituration. The cells were than spun downre-suspended in serum free medium and counted. The cells were alsostained with trypan blue to determine the rate of viable cells.

[0394] (d) Cryopreservation of Spheres

[0395] Spheres of precursors were transferred into a 1.2 ml cryo-vial(Nalge Nunc Naperville, Ill.) containing 0.5-1 ml of pre-cooled (4° C.)freezing medium (90% serum free medium (as above) and 10% DMSO (Sigma)).The vials were slowly cooled (˜1° C. min) in a freezing container(Nalgene, Nalge Nunc Napervville, Ill.) to −80° C. and then plunged intoand stored in liquid nitrogen. The vials were rapidly thawed in a waterbath at 37° C. The freezing medium was gradually diluted with 10 mlserum free culture medium and the spheres were transferred to freshserum free medium.

[0396] 7. Characterization of the Progenitor Cells in the Spheres

[0397] (a) Immunohistochemistry Studies

[0398] In general, for the immunophenotyping of spheres, disaggregatedprogenitor cells and differentiated cells, fixation with 4%paraformaldehyde for 20 minutes at room temperature was used unlessotherwise specified. It was followed by blocking and permeabilizationwith 0.2% Triton X (Sigma) and 5% heat inactivated goat serum (Dako) inPBS for one hour. Samples were incubated with the primary antibodies atroom temperature for 30 minutes, washed, incubated with the secondaryantibodies for the same time, counter-stained and mounted withVectashield mounting solution with DAPI (Vector Laboratories,Burlingame, Calif.). Primary antibodies localisation was performed byusing swine anti-rabbit and goat anti-mouse immunoglobulins conjugatedto fluorescein isothiocyanate (Dako; 1:20), and goat anti mouse lgMconjugated to Texas Red (Jackson Lab. West Grove, Pa.: 1:50). Propercontrols for primary and secondary antibodies revealed neithernon-specific staining nor antibody cross reactivity.

[0399] The spheres were plated on coverslips coated with poly-D-lysine(30-70 kDa, Sigma) and laminin (Sigma), fixed after 4 hours and examinedby indirect immunofluorescence analysis for the expression of N-CAM(acetone fixation without permeabilization, mouse monoclonal antibodyUJ13a from Dako, Carpinteria, Calif.; 1:10 and anti polysialylatedN-CAM, clone MenB, kind gift of G. Rougon; 1:50)), A2B5 (4%paraformaldehyde fixation, mouse monoclonal antibody clone 105 fromATCC, 1:20), nestin (4% paraformaldehyde fixation, rabbit antiserum akind gift of Dr. Ron McKay; 1:25) and vimentin (methanol fixationwithout permeabilization , mouse monoclonal antibody Vim3B4 from RocheDiagnostics Australia, Castle Hill, NSW; 1:20).

[0400] To evaluate the proportion of cells that expressed N-CAM, A2B5,nestin and vimentin, spheres that were cultivated 6-18 weeks weredissaggregated into single cells either by mechanical tituration in PBSwithout calcium and magnesium or by enzymatic (papain, WortiningtonBiochemical Co, NJ) digestion that was followed by tituration. The cellswere than plated on coverslips coated with poly-D-lysine and lamininfixed after 24 hours and examined by indirect immunofluorescenceanalysis for expression of N-CAM A2B5, nestin and vimentin. DAPIcounterstain of the cell nuclei assisted in identifying individualcells. Two hundred cells were scored within random fields (at ×400) forthe expression of each marker and the scoring was repeated at least 3times for each marker. Three progenitor cell lines derived fromdifferentiating colonies and two lines that were derived directly fromundifferentiated cells were evaluated.

[0401] To examine the expression of endodermal markers, spheres wereplated on coverslips coated with poly-D-lysine and fibronectin (Sigma, 5mcg/mk), cultured 4 weeks in the absence of growth factors and examinedby indirect immunofluorescence analysis for the expression of lowmolecular weight (LMW) cytokeratin (4% paraformaldehyde fixation, mousemonoclonal antibody from Beckton Dickinson, San Jose, Calif.) andlaminin (4% paraformaldehyde fixation, mouse monoclonal antibody, 1:500dilution, from Sigma).

[0402] (b) RT-PCR

[0403] Rt PCR was used to study the expression of nestin, thetranscription factor PAX-6, oct4, CD-34, FLK-1, AC-133, ultra highsulfur keratin, amylase, (α1 anti trypsin, transferrin HNF-3α, andalfafetoprotein (AFP), in the spheres.

[0404] Expression of the endodermal markers HNF-3α, AFP and transferinwas also studied in differentiated spheres that were plated onpoly-D-lysine (30-70 kDa) and Fibronectin (Sigma, 5 mcg/mk) or laminin(Sigma), cultured in the same serum free medium supplemented with growthfactors for two weeks and then further cultured two weeks without growthfactors supplementation.

[0405] The mRNA was isolated on magnetic beads (Dynal AS, Oslo)following cell lysis according to the manufacturer's instructions, andsolid-phase first strand cDNA synthesis was performed using SuperscriptII reverse transcriptase (Gibco, Gaithersburg, Md.).

[0406] Alternatively, total RNA was isolated by using the RNA STAT-60™kit (Tel-Test Inc, Friendswood, Tex.) and first strand cDNA synthesiswas performed using Superscript II reverse transcriptase or SuperScriptFirst Strand Synthesis System (Gibco, Gaithersburg, Md.) according tothe manufacturers' instructions.

[0407] The PCR reaction was carried out according to van Eijk et al.(1999), using the solid phase cDNA as template and Taq polymerase(Pharmacia Biotech, Hong Kong). Alternatively, the PCR reaction mixturecontained 1×PCR buffer, each primer at 0.2 μM, 0.2 μM dNTPs, 1u Taq DNAPolymerase (Gibco) or 1 u Tf1 DNA Polymerase (Promega, Madison, Wis.)and 1.5 μM Mg⁺² in a final volume of 25 μl. As a control for mRNAquality, beta-actin transcripts were assayed using the same RT-PCR. PCRprimers were synthesized by Besatec or Pacific Oligos (Adelaide,Australia). The following primers were used: Product Gene Primer sizePAX-6 Forward: 5′AACAGACAGAGCCCTCACAAACA3′ 274 bp Reverse:5′CGGGAACTTGAACTGGAACTGAC3′ Nestin Forward: 5′CAGCTGGCGCACCTCAAGATG3′208 bp Reverse: 5′AGGGAAGTTGGGCTCAGGACTGG3′ Oct-4 Forward:5′-CGTTCTCTTTGGAAAGGTGTTC 320 bp Reverse: 3′-ACACTCGGACCACGTCTTTC beta-Forward: 5′-CGCACCACTGGCATTGTCAT-3′ 200 bp actin Reverse:5′-TTCTCCTTGATGTCACGCAC-3′ beta- Forward: 5′-TCACCACCACGGCCGAGCG-3′ 291bp actin Reverse: 5′-TCTCCTTCTGCATCCTGTCG-3′ CD-34 Forward:5′-TGAAGCCTAGCCTGTCACCT-3′ 200 bp Reverse: 5′-CGCACAGCTGGAGGTCTTAT-3′FLK-1 Forward: 5′-GGTATTGGOAGTTGGAGGAA-3′ 199 bp Reverse:5′-ACATTTGCCGCTTGGATAAC-3′ AC-133 Forward: 5′-CAGTCTGACCAGCGTGAAAA-3′200 bp Reverse: 5′-GGCCATCCAAATCTGTCCTA-3′ Hnf-3α Forward:5′-GAGTTTACAGGCTTGTGGCA-3′ 390 bp Reverse: 5′-GAGGGCAATTCCTGAGGATT-3′AFP Forward: 5′-CCATGTACATGAGCACTGTTG-3′ 340 bp Reverse:5′-CTCCAATAACTCCTGCTATCC-3′ transferin Forward:5′-CTGACCTCACCTGGGACAAT-3′ 367 bp Reverse: 5′-CCATCAAGGGACAGCAACTC-3′Amylase Forward: 5′-GCTGGGCTCAGTATTCCCCAAATAC-3′ 490 bp Reverse:5′-GACGACAATCTCTGACCTGAGTAGC-3′ α1 anti Forward:5′-AGACCCTTTGAAGTCAAGGACACCG-3′ 360 bp trypsin Reverse:5′-CCATTGCTGAAGACCTTAGTGATGC-3′ Keratin Forward:5′-AGGAAATCATCTCAGGAGGAAGGGC-3′ 780 bp Reverse:5′-AAAGCACAGATCTTCGGGAGCTACC-3′

[0408] Amplification conditions were as follows: 94° C. for 4 minfollowed by 40 cycles of 94° C. for 15 sec, 55° C. for 30 sec, 72° C.for 45 sec and extension at 72° C. for 7 min.

[0409] Products were analysed on a 1.5% or a 2% agarose gel andvisualised by ethidium bromide staining.

[0410] (c) Neuronal Differentiation Studies

[0411] In general, differentiation was induced by plating the spheres onan appropriate substrate (poly-D-lysine, 30-70 kDa, and laminin, Sigma)combined with the removal of growth factors.

[0412] Two protocols were most commonly used: In the first one,differentiation was induced by plating the spheres on coverslips coatedwith poly-D-lysine and laminin in the same serum free medium detailedabove without growth factors supplementation. The cells in the sphereswere allowed to spread and differentiate for 2-3 weeks and the mediumwas changed every 3-5 days. In some of the experiments, starting fromthe sixth day after plating, the medium was supplemented with all transretinoic acid (Sigma, 10-6M).

[0413] In the second protocol, the spheres were plated on coverslipscoated with poly-D-lysine and laminin in serum free growth mediumsupplemented with growth factors. After 5-6 days the supplementation ofgrowth factors was withdrawn and all trans retinoic acid (Sigma, 10-6M)was added to the medium. The cells were further cultured for 1-2 weeks.The medium was changed every 5 days.

[0414] (d) Characterization of Differentiated Neuronal Cells

[0415] Differentiated cells growing out from the spheres were analysed2-3 weeks after plating by indirect immunofluorescence for theexpression of the following markers: 200 kDa neurofilament protein (4%paraformaldehyde fixation, mouse monoclonal antibody RT97 fromNovocastra, Newcastle, UK), 160 kDa neurofilament protein (methanolfixation without permeabilization, mouse monoclonal NN18 from Chemicon,Temecula, Calif.; 1:50 ) 68 kDa neurofilament protein (100% ethanol,Amersham, Amersham U.K), 70 kDa neurofilament protein (Chemicon; 1:100),MAP2 a,b (4% paraformaldehyde fixation, mouse monoclonal AP20 fromNeomarkers, Union City, Calif.; 1:100), glutamate (1% paraformaldehydeand 1% glutaraldehyde, rabbit antiserum from Sigma; 1:1000),synaptophysin (4% paraformaldehyde, mouse monoclonal SY38 from Dako;1:50), tyrosine hydroxylase (4% paraformaldehyde, mouse monoclonal,Sigma), serotonin (Sigma; 1:1000), glutamic acid decarboxylase (GAD, 1%paraformaldehyde, 1% glutaraldehyde, rabbit antiserum from Chemicon,Temecula, Calif.; 1:200), GABA (4% paraformaldehyde, Sigma; 1:1000),β-tubulin (4% paraformaldehyde, mouse monoclonal TUB 2.1 from Sigma) andβ-tubulin III (4% paraformaldehyde mouse monoclonal SDL.3D10 from Sigma;1:150).

[0416] Differentiated cells were also analysed 2-3 weeks after platingby RT-PCR for the expression of β-actin, glutamic acid decarboxylase(primers, Vescovi et al., 1999) GABA_(A) receptor subunit α2 (primers,Neelands et al., 1998), neuron-specific enolase, neurofilament mediumsize chain (NF-M) (primers, Kukekov et al 1999). mRNA preparation andthe RT-PCR reaction were carried out as described above.

[0417] (e) Glial Differentiation Studies

[0418] At the time of routine passage spheres were subcultured intoserum free medium (as detailed above) supplemented with platelet derivedgrowth factor (recombinant human PDGF-AA, Peprotech Inc 20 ng/ml) andbFGF (Gibco, 20 ng/ml). Fifty percent of the medium was replaced byfresh medium every 3 days. After culture for 6 days the spheres wereplated on coverslips coated with poly-D-lysine and laminin in the sameserum free medium without growth factors supplementation. The cells inthe spheres were allowed to spread and differentiate for 10-12 days andthe medium was changed every 3-5 days. An alternative protocol was usedin some of the experiments. In these experiments the spheres werecultured in serum free medium (as detailed above) supplemented withPDGF-AA, (20 ng/ml) and bFGF (20 ng/ml) for three weeks. The sphereswere then plated on coverslips coated with poly-D-lysine and Fibronectin(Sigma, 5 mcg/mk). They were cultured for a week in the serum freemedium supplemented with PDGF-AA, (20 ng/ml), bFGF (20 ng/ml) and T3 (30nM). The growth factors were then removed from the medium and the cellswere further cultured for another 1-2 weeks in the presence of T3 only.Fifty percent of the medium was replaced by fresh medium every 3 days.In an alternative approach, spheres that were propagated in the presenceof EGF and bFGF were plated on coverslips coated with poly-D-lysine andFibronectin (Sigma, 5 mcg/mk). EGF was removed from the medium and thespheres were cultured in the presence of T3 (30 nM) and bFGF (20 ng/ml)for a week. The cells were then further cultured for another 3-4 weeksin the presence of T3 and PDGF-AA (20 ng/ml).

[0419] Oligodendrocyte were identified by indirect immunofluorescencefor the expression of the marker O4. The cells were first incubated withthe primary (Anti oligodendrocyte marker O4, mouse monoclonal IgM fromChemicon lnt. Inc. Temecula, Calif.; 1:10) and secondary FITC orrhodamine conjugated antibodies and were then fixed with 4%paraformaldehyde.

[0420] To demonstrate differentiation into astrocyte, spheres that havebeen propagated in the presence of b-FGF and EGF were plated on coverslips coated with poly-D-lysine and fibronectin or laminin and furthercultured for 6 days in serum free medium without growth factorssupplementation.

[0421] Alternatively, spheres were propagated in the presence of PDGF-AAand bFGF for 6 weeks and were then plated on cover slips coated withpoly-D-lysine and fibronectin . They were allowed to spread into amonolayer in the presence of the above growth factors for a week. Thecells were then further cultured for another week in the presence ofeither T3 or the combination of T3 and PDGF-AA.

[0422] Following this protocols, differentiation into astrocytes wasdemonstrated by Indirect immunofluorescence for the expression of glialfibrillary acidic protein (GFAP) ( 4% paraformaldehyde fixation, rabbitanti cow from Dako; 1:50)

[0423] Differentiation into astrocyte and oligodendrocyte cells was alsoconfirmed at the mRNA level. To induce differentiation to theselineages, spheres were plated on poly-D-lysine and fibronectin andcultured for 2 weeks in the serum free medium supplemented with EGF,bFGF and PDGF-AA. The differentiating spheres were then further culturedtwo weeks without growth factors and in the presence of T3. RT-PCR wasused as above to demonstrate the expression of GFAP and the plp gene.GFAP transcripts were assayed using the following primers:5′-TCATCGCTCAGGAGGTCCTT-3′ (forward) and 5′-CTGTTGCCAGAGATGGAGGTT-3′(reverse), band size 383 bp. The primers for the analysis of plp geneexpression were 5′-CCATGCCTTCCAGTATGTCATC-3′ (forward) and5′-GTGGTCCAGGTGTTGAAGTAAATGT-3′ (reverse). The plp gene encodes theproteolipid protein and its alternatively spliced product DM-20 whichare major proteins of brain myelin. The expected band size for plp is354 bp and for DM-20 is 249 bp (Kukekov et al., 1999). As a control formRNA quality, beta-actin transcripts were assayed using the same primersas above. Products were analysed on a 2% agarose gel and visualised byethidium bromide staining.

[0424] (f) Transplantation Studies

[0425] Spheres pre-labeled by the addition of 50 μM BrdU (Sigma) to theculture medium for 10 days, were dis-aggregated into small clumps eithermechanically or by enzymatic (papain, Wortinington Biochemical Co, NJ)digestion that was followed by tituration. Approximately 50,000 cells(in 2 μl PBS) were injected into the lateral ventricles of newborn(first day after birth) mice (Sabra mice, Harlan, Jerusalem) and rats(Sabra) by using a micro-glass pipette (300 micron outer diameter)connected to a micro-injector (Narishige, Japan). The glass pipette wascovered by a plastic sleeve that limited the depth of penetration intothe host nervous system. At 4-6 weeks of age, recipients wereanesthetized and perfused with 4% paraformaldehyde in PBS.

[0426] Detection and Characterization of Donor Human Neural Progenitorsin vivo

[0427] Serial 7-micrometer frozen sections were examined byimmunostaining after post fixation with acetone or with 4%paraformaldehyde and histologically after hematoxylin and eosinstaining. The transplanted cells were detected by immunuostaining withantibodies for BrdU (Dako, 1:20, following specific instructions by themanufacturer), anti human specific ribonuclear protein antibody(Chemicon; 1:20) and anti human specific mitochondrial antibody(Chemicon; 1:20). BrdU antibody was detected by using theperoxidase-conjugated Vectastain kit (Vector Burlingame, Calif.),developed with Diaminobenzadine (DAB) or by using goat anti mouse IgGconjugated to Alexa 488 (Jackson; 1:100). Anti ribonuclear protein andmitochondrial antibodies were detected with goat anti mouse IgMconjugated to Cy5 and goat anti mouse IgG conjugated to Alexa488,respectively (Jackson; 1:100). Glial cell type identity of transplantedcells was established by double staining for BrdU and GFAP (Dako; 1:100)for astrocytes, CNPase (Sigma; 1:100) and NG2 (Chemicon; 1:100) for theoligodendrocyte lineage. Neurons were detected by immunostaining withhuman specific anti neurofilament light chain (Chemicon; 1:100) and antiβIII-tubulin (antibody as detailed above; 1:100). Goat anti rabbitconjugated to Cy5 (Jackson; 1:100) and goat anti mouse IgG conjugated toAlexa488 (Jackson; 1:100) were used for detection of primary antibodies.In cases where the lineage specific antibody was a mouse monoclonal IgG,double labeling with BrdU was performed sequentially, by firstcompleting immunohistochemistry for BrdU and then performingimmunofluorescent staining for the lineage marker (i.e. CNPase orβIII-tubulin). Images were taken by a confocal microscope (Zeiss), usingchannels for Alexa488 fluorescence, Cy5 fluorescence and Nomarskyoptics.

EXAMPLES Example 1 Derivation of Cell Lines HES-1 and HES-2

[0428] The outer trophectoderm layer was removed from four blastocystsby immunosurgery to isolate inner cell masses (ICM), which were thenplated onto a feeder layer of mouse embryo fibroblasts (FIG. 1A). Withinseveral days, groups of small, tightly packed cells had begun toproliferate from two of the four ICM. The small cells were mechanicallydissociated from outgrowths of differentiated cells, and followingreplating they gave rise to flat colonies of cells with themorphological appearance of human EC or primate ES cells (FIG. 1B, Cstem cell colonies). These colonies were further propagated bymechanically disaggregation to clumps which were replated onto freshfeeder cell layers. Growth from small clumps of cells (<10 cells) wasnot possible under the conditions of these cultures. Spontaneousdifferentiation, often yielding cells with the morphological appearanceof early endoderm, was frequently observed during routine passage of thecells (FIG. 1D). Differentiation occurred rapidly if the cells weredeprived of a feeder layer, even in the presence of LIF (FIG. 1E). WhileLIF was used during the early phases of the establishment of the celllines, it was subsequently found to have no effect on the growth ordifferentiation of established cultures (not shown). Cell line HES-1 hasbeen grown for 60 passages in vitro and HES-2 for 40 passages,corresponding to a minimum of approximately 360 and 90240 populationdoublings respectively, based on the average increase in colony sizeduring routine passage, and both cell lines still consist mainly ofcells with the morphology of ES cells. Both cell lines have beensuccessfully recovered from cryopreservation.

Example 2 Marker Expression and Karyotype of the Human ES Cells

[0429] Marker and karyotype analysis were performed on HES-1 at passagelevels 5-7, 14-18, 24-26 and 44-46, and on HES-2 at passage levels 6-8.ES cells contained alkaline phosphatase activity (FIG. 2A).Immunophenotyping of the ES cells was carried out using a series ofantibodies which detect cell surface carbohydrates and associatedproteins found on human EC cells. The ES cells reacted positively inindirect immunofluorescence assays with antibodies against the SSEA-4and TRA 1-60 carbohydrate epitopes, and the staining patterns weresimilar to those observed in human EC cells (FIG. 2B, C). ES cells alsoreacted with monoclonal antibody GCTM-2, which detects an epitope on theprotein core of a keratan sulphate/chondroitin sulphate pericellularmatrix proteoglycan found in human EC cells (FIG. 2D). Like human ECcells, human ES cells did not express SSEA-1, a marker for mouse EScells. Both cell lines were karyotypically normal and both were derivedfrom female blastocysts.

[0430] Oct-4 is a POU domain transcription factor whose expression islimited in the mouse to pluripotent cells, and recent results showdirectly that zygotic expression of Oct-4 is essential for establishmentof the pluripotent stem cell population of the inner cell mass. Oct-4 isalso expressed in human EC cells and its expression is down regulatedwhen these cells differentiate. Using RT-PCR to carry out mRNA analysison isolated colonies consisting mainly of stem cells, we showed thathuman ES cells also express Oct-4 (FIG. 3, lanes 2-4). The PCR productwas cloned and sequenced and shown to be identical to human Oct-4 (notshown).

Example 3 Differentiation of Human ES Cells in vitro

[0431] Both cell lines underwent spontaneous differentiation understandard culture conditions, but the process of spontaneousdifferentiation could be accelerated by suboptimal culture conditions.Cultivation to high density for extended periods (4-7 weeks) withoutreplacement of a feeder layer promoted differentiation of human EScells. In high density cultures, expression of the stem cell markerOct-4 was either undetectable or strongly down regulated relative to thelevels of the housekeeping gene beta actin (FIG. 3, lanes 5-7).Alphafetoprotein and human chorionic gonadotrophin were readily detectedby immunoassay in the supernatants of cultures grown to high density.Alphafetoprotein is a characteristic product of endoderm cells and mayreflect either extraembryonic or embryonic endodermal differentiation;the levels observed (1210-5806 ng/ml) are indicative of extensiveendoderm present. Human chorionic gonadotrophin secretion ischaracteristic of trophoblastic differentiation; the levels observed(6.4-54.6 IU/Litre) are consistent with a modest amount ofdifferentiation along this lineage.

[0432] After prolonged cultivation at high density, multicellularaggregates or vesicular structures formed above the plane of themonolayer, and among these structures clusters of cells or single cellswith elongated processes which extended out from their cell bodies,forming networks as they contacted other cells (FIG. 1F) were observed.The cells and the processes stained positively with antibodies againstneurofilament proteins and the neural cell adhesion molecule (FIG. 2Eand F). Contracting muscle was seen infrequently in the cultures. Whilecontracting muscle was a rare finding, bundles of cells which werestained positively with antibodies directed against muscle specificforms of actin, and less commonly cells containing desmin intermediatefilaments (FIG. 2G and H) were often observed. In these high densitycultures, there was no consistent pattern of structural organisationsuggestive of the formation of embryoid bodies similar to those formedin mouse ES cell aggregates or arising sporadically in marmoset ES cellcultures.

Example 4 Differentiation of Human ES Cells in Xenografts

[0433] When HES-1 or HES-2 colonies of either early passage level (6;HES 1 and 2) or late passage level (HES-1, 14 and 27) were inoculatedbeneath the testis capsule of SCID mice, testicular lesions developedand were palpable from about 5 weeks after inoculation. All micedeveloped tumours, and in most cases both testis were affected. Uponautopsy lesions consisting of cystic masses filled with pale fluid andareas of solid tissue were observed. There was no gross evidence ofmetastatic spread to other sites within the peritoneal cavity.Histological examination revealed that the lesion had displaced thenormal testis and contained solid areas of teratoma. Embryonal carcinomawas not observed in any lesion. All teratomas contained tissuerepresentative of all three germ layers. Differentiated tissues seenincluded cartilage, squamous epithelium, primitive neuroectoderm,ganglionic structures, muscle, bone, and glandular epithelium (FIG. 4).Embryoid bodies were not observed in the xenografts.

Example 5 Development, Propagation and Characterisation of Human ESCells Derived Neural Progenitor Cells

[0434] a) Derivation of Neural Progenitor Cells from Human ES Cells

[0435] Colonies of undifferentiated ES cells from the cell lines HES-1and HES-2 were continuously cultured on mouse embryonic fibroblastsfeeder layer for 2-3 weeks. At one week after passage, some spontaneousdifferentiation was usually identified by changes in cell morphology inthe center of the colonies. The process of differentiation included atthis early stage the neuroectodermal lineage as evident by theexpression of early neural markers such as nestin and PAX-6 (FIG. 19).During the next two weeks of culture, the process of differentiation wasmarkedly accelerated mainly in the center of the colonies and cells withshort processes that expressed the early neuroectodermal marker N-CAMcould be demonstrated. It appeared that the N-CAM positive cells weregrowing out from adjacent distinct areas that were comprised of smallpiled, tightly packed cells that did not express GCTM-2, a marker ofundifferentiated ES cells or the early neuroectodermal marker N-CAM(data not shown). These distinct areas had a typical uniform white grayopaque appearance under dark field stereo-microscopy (FIG. 26). Theseareas could be identified in the colonies of both cell lines from thesecond week after passage, and they became more defined from neighboringareas of the colony during the third week of culture (FIG. 26). The sizeand demarcation of these areas was enhanced if the serum containing EScell culture medium was replaced after a week or preferably after twoweeks from passage with serum free medium supplemented with EGF (20ng/ml) and FGF (20 ng/ml). The areas were large and well demarcatedsufficiently to allow mechanical removal of clumps of cells by amicropipette in 54% of the colonies cultured in serum containing medium(67/124, HES-1). Clumps were removed from differentiating colonies ofHES-1 and HES-2 and were transferred to serum free medium supplementedwith basic fibroblast growth factor (bFGF) and epidermal growth factor(EGF). At the time of isolation, the clumps were comprised mostly of alayer of the small tightly packed cells (about 100-300 cells/clump), ontop of some loosely attached larger cells, It was possible to removethese larger cells mechanically or by enzymatic digestion. Within anhour the clumps started to change their shape toward spheres and after24 hours all the clumps turned into round spheres (FIG. 5a).

[0436] During the initial two weeks in culture, some cell death wasobserved. After 7-10 days in culture, gradual increase in the size ofthe majority of the spheres was evident and most of the spheres werestill floating or loosely attached to the dish while a minority attachedand started to spread. A detailed analysis of marker expression and thegrowth and differentiation potential of the cells within the spheres wasconducted in three preparations of spheres that were separately derivedwith this approach.

[0437] In an alternative approach, somatic differentiation of ES cellsinto spheres of progenitor cells was induced by transferring clumps ofundifferentiated ES cells into serum free medium supplemented with basicfibroblast growth factor (bFGF) and epidermal growth factor (EGF).Within 24 hours the clumps have turned into spheres. Some of thesespheres were round and some had an irregular shape. After 72 hours inserum free medium 42% (10/24) of the spheres had a round symmetricalappearance (FIG. 9) and after 12 days 62.5% (15/24). Significant growthwas observed in the majority of the spheres during this early culture.It was possible to measure and calculate the average volume of the roundfloating spheres and it increased by 64% (mean growth of 15 spheres)between days 5 and 12. Two preparations of spheres that were separatelyderived with this approach were further propagated and characterized.

[0438] b) In vitro Propagation of Spheres of Progenitor Cells

[0439] After 7-10 days in culture, floating or loosely attached sphereswith a diameter of >0.5 mm were sub-cultured by mechanical dissectioninto 4 pieces, which were re-plated in fresh pre-equilibrated medium.The spheres were cultivated in this manner during a five to six monthperiod (15 passages). Although some of the spheres had an irregularshape at the initial phase of culture, the rate of round symmetricalspheres increased along propagation. In addition, while at early passagelevels the appearance (under a stereo-microscope) of the inner part ofthe spheres was irregular, it gradually turned to be uniform at moreadvanced passage levels. By passage level five (five-six weeks afterderivation) all spheres had a round symmetrical shape and a uniformappearance.

[0440] Proliferation of the cells was evaluated by determining theincrease in the number of spheres with each passage as well as measuringthe increase in the volume of the spheres along time . In general, thegrowth rate of spheres that were generated either from undifferentiatedor from differentiating ES cells had a similar pattern characterized bya more excessive growth during the first 5-6 passages. The number ofspheres increased by 126%+54% (Mean+SD, sum of results from 3 celllines) at each passage (performed every 7 days) during the first 5passages. The growth of the number of spheres with each passage was thenreduced to 10-50% per week. This growth rate was maintained forprolonged periods (4 months) (mean data from 3 cell lines). The meanvolume of spheres generated either from differentiating ES cells ordirectly from undifferentiated cells also increased by similar rates(FIGS. 16 and 17). A relatively rapid growth rate was observed duringthe first 5-6 weeks after derivation with a population doubling time ofapproximately 4.7 days. It was followed by a 10-16 week period of slowand stable cell growth with a population doubling time of approximatelytwo and a half weeks. At this point the spheres ceased to grow and theirvolume was stable or declined (FIG. 31).

[0441] Dis-aggregation of the spheres by using trypsin digestion couldbe ineffective in particular when the spheres were cultivated forprolonged periods, however it was possible to dis-aggregate them into asingle cell suspension mechanically following enzymatic digestion withpapain. A linear correlation was found between the volume of spheres andthe number of cells within the spheres at various passage levels (5-15weeks after derivation) indicating the validity of monitoring theincrement of sphere volume as an indirect indicator of cellproliferation. The coefficients that define the regression line of thiscorrelation were similar in spheres that were derived fromdifferentiating or undifferentiated ES cells. Most of the cells (>90%)were viable following the dissagregation procedure (FIG. 10, FIG. 18).

[0442] Given the growth rate of the spheres with each passage and thenumber of cells (20,000, FIG. 10, 18) per averaged size sphere (0.1 mm³based on the mean diameter ±S.D. of 24 spheres 7 days after passage 5,0.59±0.14 mm), it was calculated that 10 clumps of ES cells may generatewithin 10 passages 2500 spheres containing 50×10⁶ cells. A cumulativegrowth curve is presented in FIG. 31 to illustrate this potential forsignificant expansion.

[0443] Spheres that were cultured in the serum free growth medium(supplemented with growth factors) for prolonged periods (3 weeks)without passage, tended to attach to the tissue culture plastic andgradually spread as a monolayer of cells. The cells in the monolayer hada uniform appearance of neural progenitors and a high mitotic activitywas evident (FIG. 7).

[0444] It was possible to recover the spheres from cryopreservation.

[0445] c) Characterization of the Progenitor Cells within the Spheres

[0446] Cells in the spheres that were produced either fromdifferentiating ES cell colonies or directly from undifferentiated EScells expressed markers of primitive neuroectoderm and neural progenitorcells, such as polysialylated N-CAM (FIG. 5b, 11, 12), A2B5, theintermediate filament proteins nestin (immunostaining, FIG. 5c and 13;RT-PCR, FIG. 3b and FIG. 19) and vimentin (FIG. 5d and 15), and thetranscription factor Pax-6 (FIG. 3b and FIG. 19). The expression ofthese markers was maintained along prolonged cultivation (18 weeks). Thetranscriptional factor oct4 was not expressed by the cells in thespheres indicating that undifferentiated human ES cells were not presentwithin the spheres (FIG. 19).

[0447] To evaluate the proportion of cells in the spheres that expressedN-CAM, A2B5, nestin and vimentin, spheres that were cultivated at least6 weeks (and up to 18 weeks) were disaggregated to single cellsuspension. The resulting single cells were plated on substrate ingrowth medium. Twenty four hours after plating, an average of 99±1.6%(n=11 experiments) and 95.5% (95.7%-96.7%, n=6) of the cells fromspheres generated form differentiating (three progenitor cell lines) andundifferentiated ES cells (two progenitor cell lines) respectively weredecorated with the antibody against N-CAM. The average proportion ofcells that were positively stained for nestin, A2B5, and vimentin was97±2.3% (n=10 experiments) 89.5% (n=2 experiments) and 67±16.8% (n=9experiments) respectively in spheres that were established fromdifferentiating colonies. Nestin and vimentin were expressed by 66.8%(48.5%-100%, n=5) and 58% (41.6%-76.5%, n=5) of the cells thatoriginated from spheres that were generated from undifferentiated cells.These proportions were stable during prolonged cultivation (18 weeks).

[0448] The high proportion of cells that expressed N-CAM indicate thatthe spheres from both sources were comprised of a highly enrichedpreparation of neural progenitor cells. An extremely high proportion ofcells from spheres that were derived from differentiating ES coloniesalso expressed the neural progenitor markers nestin and A2B5. Theproportion of cells expressing nestin was less extensive in spheres thatoriginated from undifferentiated ES cells. The high proportion of cellsthat expressed these markers was stable along prolonged cultivation.

[0449] To determine whether cells from other lineages were presentwithin the spheres, the expression of markers of endoderm, epidermis andmesoderm was examined by RT-PCR and immunohistochemistry.

[0450] There was no evidence for the expression of markers of theextraembryonic endodermal lineage (HNF-3α, AFP, RT-PCR, FIG. 24,) bycells of spheres that were derived by either methods. Moreover, theexpression of markers of the endodermal lineage was also not detected inspheres that were derived from differentiating colonies and that wereinduced to differentiate by plating on an appropriate substrate andculturing in the absence of growth factors for 4 weeks (HNF-3α, AFP,were evaluated by RT-PCR; LMW cytokeratin and laminin were_evaluated byimmunohistochemistry). ES cell colonies that were induced todifferentiate by prolonged culture (3-4 weeks) expressed all of theabove markers and served as positive controls.

[0451] However, expression of markers of mesodermal precursors (FLK-1and CD-34) was demonstrated in the spheres that were produced by eithermethod (FIG. 24). In addition, transcripts of markers of embryonicendoderm (α1 anti trypsin, transferrin and amylase), epidermis (keratin)and the hematopoietic lineage (Ac-133) were demonstrated in spheres thatwere derived from differentiating ES colonies (FIG. 32).

[0452] It may be concluded that the spheres were comprised of a highlyenriched population of neural progenitors (>95%) and probably no cellsfrom the extraembryonic endodermal lineage. The expression oftranscripts of non-neural markers may indicate the presence of a minutepopulation of cells from other lineages within the spheres.Alternatively it may be that the primitive neural precursors within thespheres express these markers. The expression of the hematopoieticmarker AC-133 (Uchida et al., 2000) that was recently demonstrated inneural stem cells derived from human fetal brains support thispossibility. In addition, expression of non-neural markers by the neuralprogenitors may be in line with the recently reported broad potential ofneural stem cells to trans-differentiate into a variety of tissues(Bjornson et al., 1999; Clarke et al., 2000).

[0453] (d) In vitro Neuronal Differentiation

[0454] When plated on poly-D-lysine and laminin, spheres that wereproduced either from differentiating ES cell colonies or fromundifferentiated ES cells attached, and differentiated cells grew outonto the monolayer from them.

[0455] When the bFGF and EGF were removed at the time of plating,differentiating cells gradually spread from them in a radial fashion(FIG. 6a, FIG. 33) If the growth factors were removed only after 1-2weeks, a more extensive spreading of cells with processes, which formeda monolayer was evident (FIG. 8). Two to three weeks after plating, thedifferentiated cells originating from spheres derived by either methodsdisplayed morphology and expression of structural markers characteristicof neurons, such as β-tubulin (FIG. 6h), β-tubulin III (FIG. 27c, the200 kDa neurofilament (FIG. 6b) and 68 kDA neurofilament proteins andneuron specific enolase (NSE, FIG. 34). Moreover, differentiated cellsoriginating from spheres derived by either methods expressed markers ofmature neurons such as the 160 kDa neurofilament protein (FIG. 6c, FIG.27a, FIG. 34), Map-2a,b (FIG. 6d, FIG. 27b) and synaptophysin (FIG. 6F),Furthermore, the cultures contained cells which synthesised glutamate(FIG. 6e), serotonin (FIG. 35a), GABA (FIG. 35b), expressed the ratelimiting enzyme in GABA biosynthesis( glutamic acid decarboxylase, FIG.3c and 6 g), expressed the enzyme tyrosine hydroxylase (FIG. 28) andreceptor subunits characteristic of GABA minergic neurons (GABAα2, FIG.3d).

[0456] e) In vitro Glial Differentiation

[0457] Differentiation into both astrocyte cells and oligodendrocytecells was observed with spheres that were produced either fromdifferentiating ES cells or from undifferentiated cells.

[0458] While differentiation at a low scale toward glial cells wasobserved upon withdrawal of growth factors and plating on poly-D-lysineand laminin, various protocols were developed to enhance thedifferentiation toward this lineage. These protocols were all based onplating the neural progenitor cells on poly-D-lysin and fibronectin,which significantly enhanced the differentiation toward glial cells, andsupplementation of the medium with PDGF-AA that promotes glialprogenitor cell proliferation and T3 that induces maturation ofoligodendrocytes precursors.

[0459] Differentiation into the astrocyte glial lineage was demonstratedby indirect immunofluorescent staining for GFAP. Few positive cells wereoccasionally demonstrated when differentiation was induced by withdrawalof growth factors and plating on poly-D-lysin and laminin. However,differentiation into astrocytes was significantly enhanced when thespheres were allowed to differentiate on poly-D-lysin and fibronectin.Moreover, differentiation into astrocytes was highly abundant after thefollowing protocol. The spheres were first propagated six weeks in thepresence of PDGF-AA and bFGF and were then plated on poly-D-lysine andfibronectin. They were allowed to spread for a week into a monolayer inthe presence of the above growth factors. The differentiating cells werethen further cultured for another week in the presence of T3 and PDGF-AAfollowed by another 1-2 weeks of culture either with T3 or thecombination of T3 and PDGF-AA (FIG. 20).

[0460] To promote differentiation towards oligodendrocyte, spheres wereinitially cultured for 6 days in serum free medium supplemented withPDGF-AA (20 ng/ml) and bFGF (20 ng/ml) and were then plated oncoverslips coated with poly-D-lysine and laminin in the same mediumwithout growth factors supplementation. The cells in the spheres wereallowed to spread and differentiate for 10-12 days. Small cellsdecorated with the antibody O4 could be demonstrated at that timeindicating differentiation into oligodendrocyte progenitors.

[0461] It was also possible to promote the differentiation intooligodendrocyte progenitors by incubation of the spheres in the presenceof PDGF and basic FGF for 3 weeks followed by plating on poly lysine andfibronectin and culture for a week in the presence of the growth factorsand T3 followed by 1-2 weeks culture in the presence of T3 withoutgrowth factors supplementation (FIG. 14). Alternatively, spheres thatwere propagated in the presence of bFGF and EGF were plated on polylysine and fibronectin and cultured for a week in the presence of bFGFand T3. The cells were then further cultured in the presence of PDGF andT3 for 3-4 weeks.

[0462] Differentiation into astrocyte and oligodendrocyte cells wasfurther confirmed at the mRNA level. Spheres were plated onpoly-D-lysine and fibronectin and cultured for 2 weeks in the serum freemedium supplemented with EGF, bFGF and PDGF-AA. The differentiatingspheres were then further cultured two weeks without growth factors inthe presence of T3. The expression of GFAP was demonstrated by RT-PCRindicating and confirming the presence of astrocyte cells (FIG. 25, FIG.34). The expression of myelin basic protein (MBP) and the plp gene wasused as markers of differentiation into oligodendrocyte cells. The plpgene encodes the proteolipid protein and its alternatively splicedproduct DM-20, which are major proteins of brain myelin.

[0463] RT-PCR analysis of the differentiated spheres demonstrated MBPand bothdm-20 and plp transcripts indicating that differentiation intooligodendrocyte has occurred (FIG. 25, FIG. 34).

[0464] f) Transplantation of Neural Spheres

[0465] To explore the developmental potential of the human ES-derivedneural progenitors in vivo, and to reveal whether the human precursorscan respond to positional cues, migrate along established host braintracts, differentiate into neural cell types according to a givenregion's stage of development and participate in the development andhistogenesis of a living host, dis-aggregated spheres were implantedinto the lateral cerebral ventricles of newborn mice. Transplantationwas performed 9-15 weeks after derivation of the neural spheres. Priorto transplantation, the neural progenitors were labeled with BrdU tofacilitate their identification in the host brain. Histological andimmunochemical evaluation of serial brain sections was performed mainly4-6 weeks after transplantation. The transplanted cells were identifiedby their immunoreactivity with anti BrdU antibodies (FIG. 21 and 36E).Numerous BrdU+ cells were found in 9 out of 14 transplanted animals andsuccessful engraftment was documented with donor cells from the threeneural progenitor clones. The human identity of cells that weredecorated with anti BrdU was confirmed by double labeling demonstratingimmunoreactivity with both anti BrdU and anti human specificribonucleoprotein antibodies (FIG. 37B). The identity of thetransplanted human cells was also confirmed by immunofluorescentstaining with a human-specific anti-mitochondrial antibody (FIG. 37A).

[0466] Transplanted newborns that were examined during the firstpostnatal week exhibited clusters of donor cells lining the ventricularwall (FIG. 36A). At 4-6 weeks following implantation, human cells hadleft the ventricles and migrated in large numbers mainly as individualcells into the host brain parenchyma. The human cells demonstrated awidespread distribution in various regions of the host brain includingperiventricular areas (FIG. 22), the entire length of the corpuscallosum, fimbria, internal capsule, as well as into diencephalic tissuearound the 3rd ventricle (FIG. 36B, D). Transplanted human cells alsomigrated anteriorily from the subventricular zone along the rostralmigratory stream (FIG. 23, FIG. 36C) and populated the olfactory bulb,indicating their potential to respond to local cues and migrate alongestablished host brain tracts. In addition, BrdU+ cells were found inthe dentate gyrus (not shown), where post-natal neuronogenesis is knownto occur as well.

[0467] Differentiation in vivo into the three fundamental neurallineages was demonstrated by immunochemical studies using anti humancell type specific antibodies or double labeling experiments with bothanti BrdU and anti neural cell type specific antibodies. Glialdifferentiation of the transplanted cells was abundant in theperiventricular areas that consist of white matter tracks where glialdifferentiation in the post natal period is predominant. Double labelingimmunochemical studies for BrdU and GFAP demonstrated cells that weredecorated by both antibodies indicating in-vivo differentiation intoastrocytes (FIG. 29, FIG. 37C). Transplanted cells that havedifferentiated into the oligodendroglial lineage and were reactive withanti BrdU and anti NG-2 (a marker of oligodendrocyte progenitors) oranti 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), were alsodemonstrated in these areas (FIG. 30 and FIG. 37D and E).

[0468] Neuronal differentiation of the human transplanted cells wasspecifically demonstrated in the olfactory bulb, a region whereneuronogenesis occurs after birth. Neuronal processes of transplantedcells were also detected by a human specific anti light chainneurofilament antibody in the fimbria (FIG. 37F and G).

[0469] There was no histological evidence of tumor formation in any ofthe recipient animals.

[0470] Our data demonstrated that neural progenitors that were derivedfrom human ES cells in vitro could respond appropriately to normaldevelopmental cues in vivo. Following transplantation to the cerebralventricles of newborn mice, the donor cells migrated in large numbersinto the host brain parenchyma and demonstrated widespread distribution.Migration was not random and the transplanted human progenitors followedestablished brain pathways as demonstrated by their migration along theRMS, suggesting their responsiveness to host's signals. The human ESderived neural progenitors differentiated in vivo into neurons,astrocyte and oligodendrocyte cells. Differentiation into neurons wasdemonstrated only in the olfactory bulb where host differentiation intothis lineage occurs in the postnatal period, whereas differentiationinto astroglia and oligodendroglia was demonstrated in subcortical areaswhere gliogenesis predominates and neurogenesis has ended. These datademonstrate that cell fate was determined in a region specific mannerand according to a given region's stage of development. It should benoted that the ability to integrate and participate in host brainhistogenesis was maintained after prolonged proliferation of the ESderived neural progenitors in vitro.

Example 6 Cryo-preservation of Human ES Cells

[0471] Attempts to cryo-preserve human ES cells by using conventionalslow freezing protocols were associated with a very poor outcome afterthawing. Since ES cells are derived from the blastocyst and retain theirembryonic properties in culture, we have postulated thatcryopreservation by using a method which is efficient for embryos may bebeneficial. Early passage clumps of human ES cells were frozen by usingthe open pulled straw (OPS) vitrification method which was recentlyshown to be highly efficient for the cryopreservation of bovineblastocysts (Vatja et al. 1998). Both cell lines were successfullythawed and further propagated for prolonged periods. The outcome of thevitrification procedure was further studied on cell line HES-1, andrecovery of viable cells with this procedure was found to be highlyefficient. All clumps (n=25) survived the procedure and attached andgrew after thawing. Vitrification was associated with some cell death asevidenced by the reduced size of colonies originating from vitrifiedclumps two days after thawing in comparison to colonies fromnon-vitrified control clumps. However, two days in culture weresufficient to overcome this cell deficit, and 9 days after plating thesize of colonies from frozen-thawed clumps exceeded that of controlcolonies at 7 days. Vitrification did not induce differentiation afterthawing. Thawed cells retained a normal karyotype and the expression ofprimate stem cell markers, and formed teratomas in SCID mice.

[0472] Finally it is to be understood that various other modificationsand/or alterations may be made without departing from the spirit of thepresent invention as outlined herein.

1. An enriched preparation of human undifferentiated embryonic stemcells wherein said cells are capable of proliferation in vitro anddifferentiation to neural progenitor cells, neuron cells and/or glialcells.
 2. The enriched preparation of human undifferentiated embryonicstem cells according to claim 1 wherein said cells maintain anundifferentiated state when cultured on a fibroblast feeder layer in theabsence of a differentiating signal.
 3. The enriched preparation ofhuman undifferentiated embryonic stem cells according to claim 1 or 2wherein said cells are capable of differentiation into neural progenitorcells.
 4. An undifferentiated human embryonic stem cell wherein the cellis capable of proliferation in vitro and differentiation to neuralprogenitor cells, neuron cells and/or glial cells and is immunoreactivewith markers for human pluripotent stem cells including SSEA-4, GCTM-2antigen, and TRA 1-60.
 5. The undifferentiated human embryonic stem cellaccording to claim 4 wherein the cell expresses Oct-4.
 6. Theundifferentiated human embryonic stem cell according to claim 5 whereinsaid cell maintains a diploid karyotype during prolonged cultivation invivo.
 7. The undifferentiated human embryonic stem cell according to anyone of claims 4 to 6 which forms tumors when injected in the testis ofimmunodeprived SCID mice.
 8. A differentiated committed human progenitorcell line capable of differentiation and propagation into mature neuronsand/or glial cells said cell line derived from undifferentiated humanembryonic stem cells.
 9. The differentiated committed human progenitorcell line according to claim 8 capable of establishing a graft in arecipient brain.
 10. The differentiated committed human progenitor cellline according to claim 9 capable of differentiating in vivo into othercell lineages including neurons and glial cells wherein the glial cellsare selected from the group including astrocytes and oligodendrocytes.11. A neural progenitor cell differentiated in vitro from anundifferentiated human embryonic stem cell.
 12. The neural progenitorcell according to claim 11 wherein said cell is capable ofproliferation.
 13. The neural progenitor cell according to claim 11wherein said cell is capable of differentiating to a mature neuron cellor glial cell.
 14. The neural progenitor cell according to claim 11wherein said cell is capable of transdifferentiation into other celllineages to generate stem cells and differentiated cells of non-neuralphenotype including hemangioblast, haematopoietic stem cells,endothelial stem cells, embryonic endoderm and ectodermal cells.
 15. Thedifferentiated neural progenitor cell according claims 8 or 11characterised by expressed markers including markers of theneuroectodermal lineage; markers of neural progenitor cells;neuro-filament proteins; monoclonal antibodies including MAP2ab;glutamate; synaptophysin; glutamic acid decarboxylase; GABA, serotonin,tyrosine hydroxylase; β-tubulin; β-tubulin III; GABA Aα2 receptor, glialfibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide3′-phosphodiesterase (CNPase), plp, DM-20, O4 and NG-2 immunostaining.16. The neural progenitor cell according to claim 15 which expressesmarkers of neuroectoderm and neural progenitor cells selected from thegroup including polysialylated N-CAM, N-CAM, A2B5, nestin, vimentin andthe transcriptional factor Pax-6, and do not express Oct-4.
 17. Theneural progenitor cell according to claim 16 wherein said cell iscapable of establishing a graft in a recipient brain.
 18. The neuralprogenitor cell according to claim 17 wherein said cell can incorporateextensively into a recipient brain.
 19. The neural progenitor cellaccording to claim 18 wherein said cell is capable of migrating alonghost brain pathways.
 20. The neural progenitor cell according to claim19 wherein said cell is capable of wide spread distribution in hostbrain.
 21. The neural progenitor cell according to claim 20 wherein saidcell is responsive to host environmental signals.
 22. The neuralprogenitor cell according to claim 21 wherein said cell differentiatesin response to local host environmental signals.
 23. The neuralprogenitor cell according to claim 22 wherein said cell is capable ofdifferentiation to progeny of neural lineages selected from the groupincluding neurons, oligodendrocyte and astrocyte in a recipient brain.24. The enriched preparation of neural progenitor cells including anenrichment of cells according to claim
 23. 25. The enriched preparationof neural progenitor cells according to claim 24 wherein said cells arecapable of prolonged undifferentiated proliferation and expansion in invitro culture.
 26. The enriched preparation of neural progenitor cellsaccording to claim 24 wherein said cells are capable of differentiationinto neurons, mature neurons and glial cells.
 27. The enrichedpreparation of neural progenitor cells according to claim 24 whereinsaid cells are capable of establishing a graft in a recipient brain inthe absence of tumors.
 28. The enriched preparation of neural progenitorcells according to claim 25 wherein said cells may be recovered fromcryopreservation.
 29. A method of preparing undifferentiated humanembryonic stem cells for differentiation into neural progenitor cells,said method including: obtaining an in vitro fertilised human embryo andgrowing the embryo to a blastocyst stage of development; removing innercells mass (ICM) cells from the embryo; culturing ICM cells underconditions which do not induce extraembryonic differentiation and celldeath, and promote proliferation of undifferentiated stem cells; andrecovering the stem cells.
 30. The method according to claim 29including culturing the ICM cells on a fibroblast feeder layer topromote proliferation of embryonic stem cells prior to recovering thestem cells from the feeder layer.
 31. The method according to claim 30wherein said fibroblasts are selected from human or mouse fibroblasts ora combination of human and mouse fibroblasts.
 32. The method accordingto claim 31 wherein the fibroblast feeder layer comprises embryonicfibroblasts.
 33. The method according to claim 32 wherein saidfibroblasts are derived from inbred 129/Sv or CBA mice or mice from across of 129Sv with C57/B16 strains.
 34. The method according to claim30 wherein said fibroblast feeder layer has a density of approximately25,000 human and 70,000 mouse cells per cm² or 75,000 to 100,000 mousecells per cm².
 35. The method according to claim 34 wherein thefibroblast feeder layer is established 6 to 48 hours prior to additionof ES or ICM cells.
 36. The method according to claim 35 wherein thefibroblast feeder cells are arrested in their growth.
 37. The methodaccording to claim 36 wherein the fibroblast feeder cells are arrestedby irradiation or treated with mitomycin C.
 38. The method according toclaims 29 further including: replating the stem cells from thefibroblast feeder layer onto another fibroblast feeder layer; andculturing the stem cells for a period sufficient to promoteproliferation of morphologically undifferentiated stem cells.
 39. Theundifferentiated human embryonic stem cell prepared by a methodaccording to claim
 38. 40. A method of inducing somatic differentiationof stem cells in vitro into progenitor cells said method comprising:obtaining undifferentiated embryonic stem cells; and providing adifferentiating signal under conditions which are non-permissive forstem cell renewal, do not kill cells and/or induces unidirectionaldifferentiation toward extraembryonic lineages.
 41. The method accordingto claim 40 wherein said undifferentiated embryonic stem cell is anundifferentiated human embryonic stem cell wherein the cell is capableof proliferation in vitro and differentiation to neural progenitorcells, neuron cells and/or glial cells and is immunoreactive withmarkers for human pluripotent stem cells including SSEA-4, GCTM-2antigen, and TRA 1-60.
 42. The method according to claim 40 wherein theconditions for inducing somatic differentiation of stem cells areselected from any one of the following including: culturing theundifferentiated stem cells for prolonged periods and at high density ona fibroblast feeder cell layer to induce differentiation; culturing theundifferentiated stem cells in serum free media; culturing theundifferentiated stem cells on a differentiation inducing fibroblastfeeder layer and wherein said fibroblast feeder layer does not induceextra embryonic differentiation and cell death; culturing to a highdensity in monolayer or on semi-permeable membranes so as to createstructures mimicing the postimplantation phase of human development; orculturing in the presence of a chemical differentiation factor selectedfrom the group including bone morphogenic protein-2 or antagoniststhereof.
 43. A differentiated progenitor cell prepared by the methodaccording to any one of claims 40 to
 42. 44. The differentiatedprogenitor cell according to claim 43 selected from the group consistingof a neural progenitor cell, mesodermal progenitor cell includinghemangioblast, hematopoietic or endothelial stem cells, endodermal andectodermal progenitors.
 45. The differentiated progenitor cell accordingto claim 44 which is a neural progenitor cell capable of differentiatinginto a neuron cell and/or a glial cell.
 46. A method of inducing somaticcells from embryonic stem cell derived somatic progenitors, said methodcomprising: obtaining a source of embryonic stem cell derived somaticprogenitors; culturing the progenitor cells on an adhesive substrate;and inducing the cells to differentiate to somatic cells underconditions which favour somatic differentiation.
 47. The methodaccording to claim 46 wherein said embryonic stem cell derived somaticprogenitor cells are grown in the presence of a serum free media andgrowth factors and are induced to differentiate by withdrawal of thegrowth factors.
 48. The method according to claim 46 wherein theembryonic stem cell-derived progenitor cell is a cell selected from thegroup consisting of a neural progenitor cell, mesodermal progenitor cellincluding hemangioblast, hematopoietic or endothelial stem cells,endodermal and ectodermal progenitors.
 49. The method according to claim46 wherein the progenitor cells are cultured on an adhesive substrateselected from poly-D-lysine and laminin or poly-D-lysine andfibronectin.
 50. The method according to claim 49 wherein the progenitorcells are cultured on poly-D-lysine and laminin.
 51. The methodaccording to claim 50 wherein the cells are further cultured in thepresence of retinoic acid.
 52. The method according to claim 47 whereinsaid somatic cells induced are neurons including mature neurons.
 53. Amature neuron cell prepared by the method according to claim 52 andcharacterised by expression of the 160 kDa neurofilament protein,MAP2ab, glutamate, synaptophysin, glutamic acid decarboxylase (GAD),GABA, tyrosine hydroxylase and serotonin.
 54. The method according toclaim 49 wherein the progenitor cells are cultured on poly-D-lysine andfibronectin.
 55. The method according to claim 54 wherein the progenitorcells are cultured before and after plating on poly-D-lysine andfibronectin in serum free medium in the presence of PDGF-AA and bFGF.56. The method according to claim 55 wherein the progenitor cells arecultured after plating in the presence of PDGF-AA, basic FGF and EGF.57. The method according to claim 56 further including culturing thesomatic progenitor cells after plating in the presence of T3.
 58. Themethod according to claim 57 wherein said somatic cells induced areglial cells including astrocyte and oligodendrocyte cells.
 59. Anoligodendrocyte cell prepared by the method according to claim 58 andcharacterized by RNA transcription of MBP, plp, dm-20 and immunostainingfor O4 and NG-2.
 60. A method of producing an enriched preparation ofhuman ES derived neural progenitor cells, said method comprising:obtaining an undifferentiated human embryonic stem cell according toclaim 39; inducing somatic differentiation of the embryonic stem cell toa neural progenitor cell by a method according to claim 40; identifyinga neural progenitor cell by expressed markers of primitive neuroectodermand neural stem cells and wherein said markers are selected from thegroup including polysialyated N-CAM, N-CAM, A2B5, intermediate filamentproteins including nestin and vimentin and the transcription factorPax-6; and culturing the neural progenitor cells to promoteproliferation and propagation.
 61. The method according to claim 60wherein the neural progenitor cells are cultured as spheres ormonolayers in serum free medium comprising DMEM/F12 supplemented withB27 and growth factors.
 62. The method according to claim 61 wherein thegrowth factors include EGF and bFGF.
 63. The method according to claim62 including further culturing to eliminate non-neural cells, inparticular extrambryonic endodermal cells, said culturing comprisingfurther selective culturing in serum free media including DMEM/F12supplemented with B27 and growth factors.
 64. The method according toclaim 63 wherein the further culturing includes the transfer ofundifferentiated ES cell clumps into serum free medium comprised ofDMEM/F12 supplemented with B27, bFGF and EGF and cultivation of theresulting neural progenitors as spheres or monolayers.
 65. A method oftransplanting ES derived neural progenitor cells in a host, said methodcomprising: obtaining a source of neural progenitor cells prepared by amethod according to claims 40; culturing the neural progenitor cells inthe presence of serum free medium supplemented with B27 and growthfactors including, EGF and bFGF; and injecting the neural progenitorcells into the nervous system of the host.
 66. The method according toclaim 65 wherein the neural progenitor cells are injected into thelateral cerebral ventricle of the nervous system.
 67. A method ofproducing a stable graft of neural cells and contributing in thehistogenesis of a living host said method comprising: transplanting ESderived neural progenitor cells into a living host by a method accordingto claim
 66. 68. A method of modifying a nervous system of a host, saidmethod comprising transplanting ES derived neural progenitor cells by amethod according to claims
 67. 69. The method according to claim 68wherein said modifying of the nervous system includes any one ofreplacing deficient neuronal or glial cell populations, restoringdeficient functions or activating regenerative and healing processes inthe nervous system to regenerate cell populations.
 70. The methodaccording to claim 68 wherein the neural progenitor cells comprisegenetically modified neural progenitor cells.
 71. The method accordingto claim 70 wherein the genetically modified neural progenitor cellsexpress specific desired genes at the target organ.
 72. A method fortreating a pathological condition of the nervous system comprisingmodifying a nervous system of a patient according to claims
 68. 73. Themethod according to claim 72 wherein the pathological condition isselected from the group including neurodegenerative disorders, mentaldisorders, vascular conditions, autoimmune disorders, congenitaldisorders, and trauma.